Significance of p53-binding protein 1 nuclear foci in uterine cervical lesions: endogenous DNA double strand breaks and genomic instability during carcinogenesis

Matsuda K, Miura S, Kurashige T, Suzuki K, Kondo H, Ihara M, Nakajima H, Masuzaki H & Nakashima M
(2011) Histopathology 59 , 441–451 Significance of p53‐binding protein 1 nuclear foci in uterine cervical lesions: endogenous DNA double strand breaks and genomic instability during carcinogenesis Aims: A defective DNA damage response can result in genomic instability (GIN) and lead to transformation to cancer. As p53‐binding protein 1 (53BP1) localizes at the sites of DNA double strand breaks (DSBs) and rapidly forms nuclear foci (NF), the presence of 53BP1 NF can be considered to be an indicator of endogenous DSBs reflecting GIN. Our aim was to analyse the presence of DSBs by immunofluorescence for 53BP1 expression in a series of cervical lesions, to evaluate the significance of GIN during carcinogenesis. Methods and results: A total of 80 archival cervical tissue samples, including 11 normal, 16 cervical intraepithelial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14 squamous cell carcinoma samples, were analysed for 53BP1 NF, human papillomavirus (HPV) infection, and p16^INK4a overexpression. The number of 53BP1 NF in cervical cells appeared to increase with progression during carcinogenesis. The distribution of 53BP1 NF was similar to that of the punctate HPV signals as determined by in‐situ hybridization and also to p16^INK4a overexpression in CIN, suggesting an association with viral infection and replication stress. Conclusions: Immunofluorescence analysis of 53BP1 expression can be a useful tool with which to estimate the level of GIN. During cervical carcinogenesis, GIN may allow further accumulation of genomic alterations, causing progression to invasive cancer.     

CD133+ cancer stem cell-like cells derived from uterine carcinosarcoma (malignant mixed Müllerian tumor)

Cancer stem cells (CSCs) that display tumor-initiating properties have recently been identified. CD133, a surface glycoprotein linked to organ-specific stem cells, has been described as a marker of CSCs in different tumor types. We herein identify and characterize CSCs in human uterine carcinosarcoma (malignant mixed Müllerian tumor), which is one of the most aggressive and therapy-resistant gynecological malignancies and is considered to be of mesodermal origin. The CD133(+) population was increased in uterine carcinosarcoma, and this population showed biphasic properties in the primary tumor. CD133(+) cells predominantly formed spheres in culture and were able to differentiate into mesenchymal lineages. CD133(+) cells were more resistant to cisplatin/paclitaxel-induced cytotoxicity in comparison with CD133(-) cells. A real-time polymerase chain reaction analysis of the genes implicated in stem cell maintenance revealed that CD133(+) cells express significantly higher levels of Oct4, Nanog, Sox2, and Bmi1 than CD133(-) cells. Moreover, CD133(+) cells showed a high expression level of Pax2 and Wnt4, which are genes essential for Müllerian duct formation. These CD133(+) cells form serially transplantable tumors in vivo and the resulting CD133(+) tumors replicated the EpCAM, vimentin, and estrogen and progesterone receptor expression of the parent tumor, indicating that CSCs likely differentiated into cells comprising the uterine carcinosarcoma tissue. Moreover, strong CD133 expression in both epithelial and mesenchymal elements in primary tumor demonstrated significant prognostic value. These findings suggest that CD133(+) cells have the characteristics of CSCs and Müllerian mesenchymal progenitors.     

Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries-Argentina, Australia, Canada, France, and Japan-were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738-740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world.     

Clonality analysis of follicular lymphoma using laser capture microdissection method

Whether a common and a single clone present, or not, among follicles of follicular lymphoma (FL) was examined in 12 cases with FL. Histologic grade was I in 6 cases, II in 3, and III in 3. DNA was selectively extracted from the neoplastic follicles of paraffin-embedded samples with use of laser capture microdissection method, and used for PCR-based analysis of rearrangement of immunoglobulin heavy chain variable region gene. Three different follicles in each case of FL were microdissected. Semi-nested PCR was performed using two sets of primers (Fr2A and Fr3A). In PCR with Fr2A primers, nine of 12 cases showed a common band among neoplastic follicles. The remaining three cases showed no PCR products. With Fr3A primers, eight of 12 cases showed a common band among follicles of the same case. The other four cases showed oligoclonal bands, among them presence of a common band was difficult to assess. Oligoclonal bands were more frequently observed in PCR with Fr3A than that with Fr2A and in grade I or II than in grade III cases. In total, 11 of 12 cases showed a common band in PCR with either Fr2A or Fr3A primers. In two cases, DNA extracted from whole section was amplified with both Fr2A and Fr3A or only Fr3A primers, showing smear or oligoclonal bands. These results showed the presence of a single clone of cells in neoplastic follicles of FL and the usefulness of PCR-based rearrangement analysis of immunoglobulin heavy chain gene combined with microdissection methods for differential diagnosis of FL from follicular hyperplasia.     

Expression of Epstein-Barr virus in Langerhans' cell histiocytosis

Langerhans' cell histiocytosis (LCH) is a proliferative histiocytic disorder of unknown etiology. We previously reported that Epstein-Barr virus (EBV) infects and proliferates in macrophages, and investigated the possibility that EBV exhibits etiologic effects in LCH. To detect EBV expression, paraffin sections from 17 LCH cases were examined by mRNA in situ hybridization for EBV BamHIW, Epstein-Barr virus nuclear antigen-2 (EBNA2), and Epstein-Barr virus-encoded small nonpolyadenylated RNA (EBER1) sequences, and by indirect immunofluorescence staining for EBNA2, latent membrane protein 1 (LMP1), and BamHIZ-coding leftward-reading frame 1 (BZLF1). To detect EBV DNA, polymerase chain reaction (PCR)-Southern blotting was used. All cases showed positive hybridization signals by BamHIW mRNA in situ hybridization. Also, 13 and 14 cases showed positive signals for EBNA2 and EBER1 RNA in situ hybridization, respectively. Furthermore, almost all cases exhibited fluorescence after immunofluorescence staining with monoclonal anti-EBNA2 and anti-BZLF1 antibodies, and 15 cases were positive after treatment with monoclonal anti-LMP1 antibody. PCR-Southern blotting detected an amplified EBER1 sequence in all 9 cases examined. EBV expression was confirmed in LCH using in situ hybridization and immunofluorescence. Furthermore, EBV DNA was also detected by PCR-Southern blotting. These positive results of BZLF1 suggest that EBV replicates in LCH tissues.     

STAT6-mediated signaling in Th2-dependent allergic asthma: critical role for the development of eosinophilia, airway hyper-responsiveness and mucus hypersecretion, distinct from its role in Th2 differentiation

When wild-type BALB/c mice were transferred with OVA-specific Th2 cells followed by OVA inhalation, a severe eosinophilia, mucus hypersecretion and airway hyper-responsiveness (AHR) was induced in parallel with a marked elevation of IL-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF). However, neither eosinophilia, AHR nor mucus hypersecretion was induced in Th2 cell-transferred STAT6-/- mice. The failure of eosinophilia was not due to the defect of Th2 cytokine production in BALF of STAT6-/- mice transferred with Th2 cells, but because of the defect of STAT6-dependent eotaxin production. Indeed, intranasal administration of eotaxin reconstituted pulmonary eosinophilia but not AHR and mucus hypersecretion in OVA-inhalated STAT6-/- mice. These results initially provided direct evidence that STAT6-dependent eotaxin production is essential for pulmonary eosinophilia. We also dissociated the role of STAT6 for eosinophilia from that for AHR and mucus hypersecretion. Thus, STAT6 also plays a critical role at late phase of Th2-dependent allergy induction.     

Induction of coronary arteritis with administration of CAWS (Candida albicans water-soluble fraction) depending on mouse strains

The intraperitoneal administration of CAWS (water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans) to mice induces coronaritis similar to Kawasaki disease. We analyzed differences in the production of cytokines involved in the occurrence of coronary arteritis among mouse strains, C3H/HeN, C57BL/6, DBA/2 and CBA/J that were injected with CAWS at 4 mg/mouse for 5 consecutive days in the first week and the fifth week of administration. The incidence of arteritis was 100% in C57BL/6, C3H/HeN and DBA/2 mice, but only 10% in CBA/J mice. The coronary arteritis observed in DBA/2 mice was the most serious, with several mice expiring during the observation period. The CAWS-sensitive strains revealed increased levels of IL-6 and IFN-gamma during the course of a specific response to CAWS by spleen cells. In contrast, IL-10 levels were observed to increase markedly in CAWS-resistant CBA/J mice, but not the CAWS-sensitive strains. However, TNF-alpha levels were more elevated only in DBA/2 mice. The difference in disease development and cytokine production strongly suggests that the genetic background of the immune response to CAWS contributes to the occurrence of coronary arteritis.     

Lymph node infarction associated with infectious mononucleosis: report of a case resembling lymph node infarction associated with malignant lymphoma

A completely infarcted lymph node should alert the pathologist to the high possibility of malignant lymphoma. The lymph node lesion of infectious mononucleosis (IM) shows marked histologic diversity and occasionally may be confused with malignant lymphoma. We report a rare case of IM showing extensive lymph node infarction whose lymph node lesion was similar to lymph node infarction associated with malignant lymphoma. This case describes a 32-year-old Japanese man who had signs and symptoms consistent with IM, which he was later proven serologically to have, but whose cervical lymph node showed extensive lymph node infarction with a thin area of granulation tissue beneath the capsule. The infarcted tissue contained numerous eosinophilic ghosts of large lymphoid cells. The thin granulation tissue was composed of numerous small lymphocytes, plasma cells, and histiocytes, in addition to large lymphoid cells including immunoblasts and granulocytes. CD20, CD3, and CD45RO immunostains revealed the mixed B- and T-cell nature of the ghosts of large lymphoid cells in the infarcted tissue as well as viable large cells in the granulation tissue. The patient was free from disease after 50 months' follow-up.     

The effect of hydrodynamics-based delivery of an IL-13-Ig fusion gene for experimental autoimmune myocarditis in rats and its possible mechanism

Interleukin (IL)-13 is a pleiotropic cytokine secreted by activated Th2 T lymphocytes. Th1 cytokines are assumed to exacerbate and Th2 cytokines to ameliorate rat experimental autoimmune myocarditis (EAM). Here, we examined the effect of IL-13 on EAM, using a hydrodynamics-based delivery of an IL-13-Ig fusion gene, as well as the possible mechanism of its effect. Rats were immunized on day 0, and IL-13-Ig-treated rats were injected with pCAGGS-IL-13-Ig, and control rats with pCAGGS-Ig, on day 1 or 7. On day 17, the IL-13-Ig gene therapy was effective in controlling EAM as monitored by a decreased heart weight/body weight ratio, by reduced myocarditis and by reduced atrial natriuretic peptide mRNA in the heart, as a heart failure marker. On the basis of IL-13 receptor mRNA expression in separated cells from EAM hearts, we proposed that IL-13-Ig target cells were CD11b(+) cells and non-cardiomyocytic noninflammatory (NCNI) cells, such as fibroblasts, smooth muscle or endothelial cells. IL-13-Ig inhibited expression of the genes for prostaglandin E synthase, cyclooxygenase-2, inducible nitric oxide synthase, IL-1beta and TNF-alpha in cultivated cells from EAM hearts, while it enhanced expression of the IL-1 receptor antagonist gene. We conclude that IL-13-Ig ameliorates EAM and suppose that its effectiveness may be due to the influence on these immunologic molecules in CD11b(+) and NCNI cells.     

Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response?

Immunological abnormalities are implicated in the pathogenesis of inflammatory bowel disease (IBD), that is, Crohn's disease and ulcerative colitis. In particular, Crohn's disease is considered to be a T helper type 1 (Th1)-shifted disease. Chemokines and their receptors are involved in various immune responses including Th1- and Th2 responses. In this study, we analyzed chemokines and their receptors by immunohistochemistry, using frozen sections derived from 33 patients with Crohn's disease and 24 with ulcerative colitis. In inflamed mucosa, small mononuclear cells predominantly expressed CCR5 and CXCR3, the receptors selectively expressed on Th1 cells, without significant differences between Crohn's disease and ulcerative colitis. We then focused on the noncaseating granulomas that are characteristic of Crohn's disease. Granuloma cells, observed in all the layers of intestinal tissues, were positive for RANTES/CCL5 protein along their cell membranes. Lymphocytes surrounding granulomas were mostly CCR5+ and CXCR3+ T cells with CD4+ and CD8+ cells at similar frequencies. Granuloma cells were positive for RANTES mRNA by in situ hybridization. By contrast, lymphoid aggregates in Crohn's disease and lymphoid follicles in the normal intestinal mucosa were characterized by abundant B cells, a predominance of CD4+ T cells over CD8+ T cells, and low frequencies of cells expressing CCR5 or CXCR3. Together with the notion that granuloma cells are possible antigen-presenting cells, our results suggest that the noncaseating granulomas could be one of the crucial sites of Th1-shifted immune responses in Crohn's disease.