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The current success of mRNA vaccines against COVID-19 has highlighted the effectiveness of mRNA and DNA vaccinations. Recently, we demonstrated that a novel needle-free pyro-drive jet injector (PJI) effectively delivers plasmid DNA into the skin, resulting in protein expression higher than that achieved with a needle syringe. Here, we used ovalbumin (OVA) as a model antigen to investigate the potential of the PJI for vaccination against cancers. Intradermal injection of OVA-expression plasmid DNA into mice using the PJI, but not a needle syringe, rapidly and greatly augmented OVA-specific CD8

Among various cytokines, interleukin (IL)-12 family cytokines have very unique characteristics in that they are composed of two distinct subunits and these subunits are shared with each other. IL-23, one of the IL-12 family cytokines, consists of p19 and p40 subunits, is mainly produced by antigen-presenting cells, and plays a critical role in the expansion and maintenance of pathogenic helper CD4+ T (Th)17 cells. Since we initially found that p19 is secreted in the culture supernatant of activated CD4+ T cells, we have further investigated the role of p19. p19 was revealed to associate with CD5 antigen-like (CD5L), which is a repressor of Th17 pathogenicity and is highly expressed in non-pathogenic Th17 cells, to form a composite p19/CD5L. This p19/CD5L was shown to activate STAT5 and enhance the differentiation into granulocyte macrophage colony-stimulating factor (GM-CSF)-producing CD4+ T cells. Both CD4+ T cell-specific conditional p19-deficient mice and complete CD5L-deficient mice showed significantly alleviated experimental autoimmune encephalomyelitis (EAE) with reduced frequency of GM-CSF+CD4+ T cells. During the course of EAE, the serum level of p19/CD5L, but not CD5L, correlated highly with the clinical symptoms. Thus, the composite p19/CD5L is a possible novel heterodimeric cytokine that contributes to EAE development with GM-CSF up-regulation.

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications.

Rationale: Since primary pure squamous cell carcinoma of the breast is a rare disease, few reports describe the characteristic findings on performing preoperative imaging that can be used to distinguish it from normal breast cancer. The rapid evolution and lack of an established method of treatment has resulted in several reports of advanced cases of primary pure squamous cell carcinoma of the breast. Patient concerns: Case 1 was a 44-year-old woman with an elastic, hard tumor in the left C region. Ultrasonographic analysis revealed a maximal 11-mm hypoechoic area. Histologically, the tumor was a well-differentiated squamous cell carcinoma with prominent keratinization, and there was prominent inflammatory cell infiltration, necrosis, and fibrosis. Case 2 was a 58-year-old woman with an elastic, hard tumor in the left C/D region. Ultrasonographic analysis revealed a maximal 31-mm hypoechoic area with partially calcified areas and a hyperechoic margin. Histologically, the tumor was a squamous cell carcinoma with prominent keratinization exhibiting an infiltrative growth pattern. The tumor had no connection to the epidermis and partially transitioned into the atypical ductal epithelium in the area surrounding the focus. Diagnoses: The patient in Case 1 was preoperatively diagnosed with T1cN0M0 Stage I cancer of the left breast, but both patients were finally diagnosed with T2N0M0 Stage IIA cancer. Interventions: Case 1: left partial mastectomy and axillary lymph node dissection were performed. The patient was administered 4 courses of FEC100 and 4 courses of DTX as postoperative adjuvant therapy. Case 2: left modified radical mastectomy and axillary lymph node dissection were performed without any postoperative adjuvant therapy. Outcomes: Case 1: no sign of relapse was observed, but the patient moved away from the area to another hospital in March 2014 and eventually died due to relapse in January 2016. Case 2: four years after surgery, no relapse has been observed. Lessons: We should always keep the presence of primary pure squamous cell carcinoma among breast cancers in mind although the crisis rate is very low. Due to its high malignancy, needle biopsy and aspiration biopsy cytology should be performed to make a definitive diagnosis.

症例は85歳，女性．平成20年6月に肛門部痛，下腹部痛を主訴に当科を紹介受診された．受診時には明らかなイレウス症状は認められなかったが，下部消化管内視鏡検査で肛門縁より5cmの位置に腸管内腔に突出するI型の隆起性病変を認め，一部表面不整を認めた．また骨盤部CT検査にて直腸内腔に重積腸管を示すmultiple concentric ring signを認めた．以上の所見より腸重積を合併した早期直腸癌の診断のもと手術を施行した．開腹後重積腸管のen-block切除を試みたが困難であり，用手的解除を施行した．解除時に一部直腸壁の損傷を認めたためHartmann手術を施行した．早期直腸癌に起因する腸重積症は稀であり，若干の文献的考察を加え報告する．

To establish a more efficient treatment for immunotherapy against solid tumors, we have evaluated the antitumor effect by coexpression of a chemokine CCL21/secondary lymphoid tissue chemokine and a costimulatory molecule LIGHT in colon carcinoma C26. C26 cells expressing either CCL21 or LIGHT exhibited a significantly reduced tumor growth in vivo, and mice inoculated with these cells showed a prolonged survival, but eventually all these mice died. In contrast, C26 cells expressing both CCL21 and LIGHT exhibited a minimal tumor growth in vivo, and all these mice survived healthily with a tumor remission and consequently acquired a strong protective immunity. A markedly increased infiltration of mature dendritic cells (DCs), and CD8(+) T cells was observed in the tumor mass, and their spleen cells showed a greatly enhanced cytotoxic T lymphocyte (CTL) activity against C26 tumor and interferon (IFN)-gamma production. Neutralization of IFN-gamma or depletion of CD8(+) or CD4(+) T cells significantly reduced the antitumor activity. These results suggest that the combined treatment with CCL21 and LIGHT is able to induce a synergistic antitumor effect to eradicate tumor completely by greatly enhancing tumor-infiltration of lymphocytes including mature DCs and CD8(+) T cells, resulting in markedly augmented CTL activity and IFN-gamma production.

Interleukin (IL)-16 is a chemoattractant cytokine for CD4(+) leukocytes. Because delayed-type hypersensitivity (DTH) reaction is mediated by T helper 1 (Th1) cells and CD4(+) T cells can be chemoattracted by IL-16, we have investigated the involvement of IL-16 in the DTH reaction. Immunohistochemical analysis revealed the IL-16 expression in infiltrating cells and epithelial cells in the DTH footpads. The IL-16 expression was also detected intracellularly in the infiltrating cells. In addition, markedly increased production of IL-16 was detected in the DTH footpad extracts, but not in the control footpad extracts, by an enzyme-linked immunosorbent assay and also by Western blot analysis. The DTH footpad extracts exhibited a strong chemoattractant activity toward splenic T cells, which was significantly inhibited by the inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in the migration assay. Furthermore, treatment of sensitized mice in vivo with the anti-IL-16 neutralizing mAb significantly suppressed the footpad swelling induced by an antigen challenge, together with decreased infiltration of leukocytes including not only CD4(+) T cells but also CD8(+) T cells and macrophages into the DTH footpads. Decreased production of macrophage inflammatory protein 1alpha was also observed in the DTH footpad extracts by the mAb treatment. These results suggest that IL-16 plays an important role in the recruitment of leukocytes-presumably including antigen-specific Th1 cells, which secrete cytokines and chemokines mediating the following hypersensitivity reaction after activation by the interaction with Langerhans cells carrying the antigen-for the elicitation of DTH response. (Blood. 2000;95:2869-2874)

We have examined the roles of gamma interferon (IFN-γ), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1 + cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-γ production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS −/− ) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS −/− mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-γ comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-γ antibody led to the progression of parasitemia and fatal outcome. CD4 −/− mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-γ production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-γ plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.

Although interleukin (IL)-12 was originally purified from an Epstein-Barr (EBV)-transformed B cell line and the high correlation of EBV infection and IL-12 expression has been suggested, no study has reported whether EBV infection is directly linked to IL-12 expression. To address this issue, we have investigated IL-12 expression in B cells during in vitro transformation with EBV. Human peripheral B cells became capable of constitutively producing p40 by in vitro transformation with EBV, coincident with the expression of latent membrane protein 1 (LMP1) of EBV. These B cells expressed p40 and p35 mRNA, and phorbol myristate acetate (PMA) stimulation strongly enhanced p40 and p70 production. Furthermore, transfection with LMP1 expression vector into a human B lymphoma cell line, Daudi, led to p40 production with nuclear factor (NF)-κB activation. These results suggest that transformation of primary B cells with EBV induces IL-12 expression potentially through LMP1 expression.

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-γ, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-α, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-γ. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-γ in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-γ production rather than the protection.

The mechanism of development of host resistance to blood-stage malarial infection was studied by use of an irradiation-induced attenuated variant, Plasmodium berghei XAT, obtained from a lethal strain, P. berghei NK65. The infection enhanced mRNA expression of interleukin (IL)-12 p40 and also of interferon (IFN)-gamma, IL-4, IL-10, and cytokine-inducible nitric oxide synthase (iNOS) in spleen. Treatment of these mice with anti-IL-12 or anti-IFN-gamma led to the progression of parasitemia and fatal outcome. Anti-IL-12 treatment significantly reduced the secretion and mRNA expression of IFN-gamma and greatly diminished the augmentation of iNOS mRNA expression. In addition, recombinant IL-12 administration delayed the onset of parasitemia because of the enhanced IFN-gamma production. These results suggest that blood-stage P. berghei XAT infection induces IL-12 production, which is important for the development of host resistance via IFN-gamma production.

Six years and 9 months ago, a 72-year-old man underwent left hepatectomy for a hilar cholangiocarcinoma(T2N0M0: Stage II A). At 3 years and 2 months after surgery, he was admitted for rectal tumor and reoperation. From surgery, it was diagnosed as peritoneal metastases of the cholangiocarcinoma, and an intraperitoneal infusion port was placed. He was treated with gemcitabine administered iv at day 1 and CDDP ip at day 8 every 3 or 4 weeks for 2 years. Then, gemcitabine was changed to docetaxel because of elevation of CA19-9. Both docetaxel iv and CDDP ip were administered for one and half years. For 3 years and 4 months, 12.2 g of CDDP, 28.0 g of gemcitabine and 920 mg of docetaxel were administered. As serum CA19-9 elevated again, he has been treated with S-1 and docetaxel ip for 3 months. This case indicates that intraperitoneal application of CDDP with systematic chemotherapy was effective for carcinomatous peritonitis without serious side effects.

Breast cancer rarely metastasizes to the pericardial cavity to cause cardiac tamponade. We have recently experienced a case of pericardial tamponade due to recurrent breast cancer. A 41-year-old woman who underwent modified radical mastectomy for a right breast cancer (T(1)N(3)M(0), Stage IIIA) 8 years and 8 months ago, was admitted for dyspnea and cough. Chest X-ray and CT scan revealed cardiomegaly and right pleural effusion, and cardiac echogram showed marked retention of pericardial effusion. A diagnosis of cardiac tamponade was made, and pericardiocentesis and thoracentesis were carried out immediately. Based on cytodiagnosis of pericardial and pleural effusion, the diagnosis was pericardial and intrapleural metastases of the breast cancer. Dyspnea was improved by pericardiocentesis and thocacentesis. Both intrapericardiac and intrathoracic instillation of CDDP prevented reaccumulation of pericardial and pleural effusion. After local chemotherapy with CDDP, systemic chemotherapy of CPT-11 was started. Thereafter the patient was discharged from the hospital and recovered her daily activities. This case indicates that intrapericardiac application of CDDP was effective for carcinomatous cardiac tamponade without serious side effects.

A 79-year-old female patient was referred to our hospital for treatment of a recurrent gallbladder cancer. Before admission, she had undergone expanded cholecystectomy and had been treated successfully with 5-FU for 3 years to suppress the tumor growth in intraperitoneal lymph nodes. The recurrence of the tumor in lymph nodes near the pancreas head was demonstrated by computer tomography. We tried a course of a combination chemotherapy consisting of CPT-11 and CDDP (40 mg CPT-11/body/day on day 1 and 10 mg CDDP/body/day on day 2-5) to reduce the size of the nodes. Then, we repeated a total of 8 courses of the therapy at 4-week intervals. The status of the nodes was not changed for a year. Then, the lymph node started to enlarge again and obstructive jaundice appeared. So, we substituted gemcitabine (1 g/body/day) for the combination chemotherapy with expandable metallic stent implantation to drain the bile. As a result, metastatic lymph nodes were reduced in size and the dilatation of the interhepatic bile duct disappeared. Thereafter, the patient was given an additional 20 courses of gemcitabine therapy at 2-week intervals as an outpatient. No change was observed in the size of the metastatic lymph nodes for a year. However, the patient died of liver metastasis 8 years after operation and 6 years after she started chemotherapy for the recurrence. She maintained a good quality of life during that time. The present case suggests that combination of chemotherapy protocols is effective for clinical management of gallbladder cancer recurrence, which is generally considered to be difficult to manage with chemotherapy.

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR γ-chain knockout (FcRγ−/−) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcγRI, FcγRII, and FcγRIII, could not induce Ab-dependent phagocytic activity. These FcRγ−/− mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-γ by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRγ−/− mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-γ−/− mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-γ production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.