Improved PCR amplification for molecular analysis using DNA from long-term preserved formalin-fixed,paraffin-embedded lung cancer tissue specimens

Archival tissue specimens are valuable resources of materials for molecular biological analyses in retrospective studies, especially for rare diseases or those associated with exposure to uncommon environmental events. Although successful amplification with PCR is essential for analysis of DNA extracted from archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens, we have often encountered problems with poor PCR amplification of target fragments. To overcome this, we examined whether heat treatment in alkaline solution could efficiently restore the PCR template activity of DNA that had already been extracted from FFPE lung cancer tissue specimens. The effect of the heat treatment was assessed by PCR for the TP53 gene and other lung cancer-related gene loci. The heat treatment of DNA samples in borate buffer resulted in successful PCR amplification of DNA fragments ranging from 91 to 152 bp. This technique for restoration of template activity of DNA for PCR amplification is very simple and economical, and requires no special apparatus, so it may be applicable for molecular analysis of DNA samples from FFPE tissue specimens at various laboratories.     

Sudden Onset of Platypnea-Orthodeoxia Syndrome Caused by Traumatic Tricuspid Regurgitation With Ruptured Chordae Tendineae After Blunt Chest Trauma

An 86-year-old man was admitted our hospital because of sudden onset of dyspnea after blunt chest trauma. Because his oxygen saturation deteriorated from 92% in the supine position to 86% in the sitting position, platypnea-orthodeoxia syndrome was suspected. Transesophageal echocardiography showed severe tricuspid regurgitation (TR) caused by anterior papillary muscle rupture. Furthermore, right-to-left shunt with TR through a patent foramen ovale (PFO) was observed. The diagnosis was therefore platypnea-orthodeoxia syndrome with right-to-left shunt through PFO with shunting exacerbated by acute severe TR after blunt chest trauma. The patient underwent urgent tricuspid valve repair and PFO closure and has remained asymptomatic postoperatively.     

IL-1 is required for tumor invasiveness and angiogenesis

Here, we describe that microenvironmental IL-1 beta and, to a lesser extent, IL-1 alpha are required for in vivo angiogenesis and invasiveness of different tumor cells. In IL-1 beta knockout (KO) mice, local tumor or lung metastases of B16 melanoma cells were not observed compared with WT mice. Angiogenesis was assessed by the recruitment of blood vessel networks into Matrigel plugs containing B16 melanoma cells; vascularization of the plugs was present in WT mice, but was absent in IL-1 beta KO mice. The addition of exogenous IL-1 into B16-containing Matrigel plugs in IL-1 beta KO mice partially restored the angiogenic response. Moreover, the incorporation of IL-1 receptor antagonist to B16-containing plugs in WT mice inhibited the ingrowth of blood vessel networks into Matrigel plugs. In IL-1 alpha KO mice, local tumor development and induction of an angiogenic response in Matrigel plugs was less pronounced than in WT mice, but significantly higher than in IL-1 beta KO mice. These effects of host-derived IL-1 alpha and IL-1 beta were not restricted to the melanoma model, but were also observed in DA/3 mammary and prostate cancer cell models. In addition to the in vivo findings, IL-1 contributed to the production of vascular endothelial cell growth factor and tumor necrosis factor in cocultures of peritoneal macrophages and tumor cells. Host-derived IL-1 seems to control tumor angiogenesis and invasiveness. Furthermore, the anti-angiogenic effects of IL-1 receptor antagonist, shown here, suggest a possible therapeutic role in cancer, in addition to its current use in rheumatoid arthritis.     

Homeostatic regulation of T follicular helper and antibody response to particle antigens by IL-1Ra of medullary sinus macrophage origin

Hepatitis B virus (HBV) vaccines are composed of surface antigen HBsAg that spontaneously assembles into subviral particles. Factors that impede its humoral immunity in 5% to 10% of vaccinees remain elusive. Here, we showed that the low-level interleukin-1 receptor antagonist (IL-1Ra) can predict antibody protection both in mice and humans. Mechanistically, murine IL-1Ra–inhibited T follicular helper (Tfh) cell expansion and subsequent germinal center (GC)-dependent humoral immunity, resulting in significantly weakened protection against the HBV challenge. Compared to soluble antigens, HBsAg particle antigen displayed a unique capture/uptake and innate immune activation, including IL-1Ra expression, preferably of medullary sinus macrophages. In humans, a unique polymorphism in the RelA/p65 binding site of IL-1Ra enhancer associated IL-1Ra levels with ethnicity-dependent vaccination outcome. Therefore, the differential IL-1Ra response to particle antigens probably creates a suppressive milieu for Tfh/GC development, and neutralization of IL-1Ra would resurrect antibody response in HBV vaccine nonresponders.

Follicular helper T (Tfh) cells are antigen-experienced CD4⁺ T cells within B cell follicles of secondary lymphoid organs, such as lymph nodes (LN), spleens, and Peyer’s patches, that constitutively express the B cell follicle homing receptor CXCR5 (1). Upon cellular interaction and cross-signaling with their cognate follicular B (FoB) cells in the presence of follicular dendritic cells (FDCs), Tfh cells trigger the formation and maintenance of germinal centers (GCs) through the expression of CD40 ligand and the secretion of IL-21 and IL-4 (2–4). Tfh-dependent paracrine activation of CD40 results in B cell survival and differentiation in the GC (5), whereas isotype class switching is believed to occur predominantly outside GCs. Therefore, Tfh cells play a critical role in mediating the selection of high-affinity B cells that differentiate either into plasma cells or memory B cells (6–11).Besides the inducible T cell costimulator (ICOS) that activates Tfh cells to secrete IL-21, other cytokines [e.g., IL-2 (12), IL-6 (13), and IL-7 (14)] also signal for Tfh cell differentiation. The role of IL-1 signaling remained puzzling until recently: Tfh cells can be primed by IL-1β, whose production is licensed by IFN-β in response to infectious agents (15). Such featured innate response of IFN-β and IL-1β relies on the activation of TLR and inflammasomes by live, but not dead, bacteria or recombinant vaccines (16, 17). Therefore, OVA antigen augments Tfh cell response in mice only when IL-1β is exogenously applied at a nonphysiological high concentration (18), whereas endogenous IL-1β/IL-1R1 signaling may not be required for antibody responses to T-dependent or -independent antigens (19–21). We reasoned that IL-1Ra (encoded by IL-1rn), which can compete with IL-1 for binding to IL-1R1 in the homeostatic inflammatory response (22–24), would intrinsically antagonize IL-1β/IL-1R1 signaling for Tfh/GC development. For example, IL-1rn^−/− mice exhibit an excessive antibody response to sheep red blood cells immunization (25, 26). A thorough investigation is required to dissect how IL-1 and IL-1Ra mutually regulate a homeostatic Tfh/GC response.LN macrophages are conventionally divided into two subtypes. Subcapsular sinus (SCS, CD169⁺F4/80⁻) macrophages are specialized antigen presenting cells that capture certain particle or opsonized antigens and display them intact for cognate recognition by FoB cells (27–30). SCS macrophages also relay immune complex to noncognate B cells for antibody affinity maturation (30). Macrophages in medullary sinus (MSM, CD169⁺F4/80⁺), in contrast, are potent in phagocytosis (31) for clearance of pathogens and particulate antigens from the lymph. It has been postulated 10 y ago that SCS may capture particle antigens, such as hepatitis B virus (HBV) vaccine, and migrate to follicles to facilitate more effective activation of B cells and FDCs (32). In this work, we found that murine antibody response inversely correlated to IL-1Ra level and clearly distinguished responders from nonresponders in volunteers receiving HBV vaccination. Further studies showed that LN macrophages subsets exhibited different capture and activation kinetics for particle and soluble antigens, and IL-1Ra expression by MSM could critically modulate IL-1R1 potentiation of Tfh cells and, hence, the specific antibody response to particle antigens. Therefore, mice lacking IL-1Ra or with IL-1Ra being neutralized yielded more robust antibody response to HBV vaccine and enables protection against chronic HBV infection.     

Lipoteichoic acid may affect the pathogenesis of bile duct damage in primary biliary cirrhosis

Aim: Intrahepatic bile ducts are the targets for inflammation in primary biliary cirrhosis (PBC), but their pathogenesis is not known. Gram-positive bacterial DNA was detected recently in gallbladder bile of PBC patients. In the present study, we assessed the possible pathological role of lipoteichoic acid (LTA), the Gram-positive bacterial cell wall component, in PBC.

Methods: Liver samples, obtained from 20 patients with PBC (stage 1–2 with CNSDC: stage 3–4 with loss of bile ducts = 10:10) and from 13 patients with chronic hepatitis due to hepatitis C virus (CH–C) with lymphocytic cholangitis, were subjected to immunohistochemical staining with polyclonal rabbit anti-LTA as the primary antibody. Serum reactivities to LTA were studied by ELISA. After 1 μg of purified LTA was placed in a 96-well microplate as an antigen, an antibody capture assay was carried out using serum samples from PBC (n = 20), CH–C (n = 13) and healthy subjects (n = 11).

Results: LTA was localized around the sites of chronic non-suppurative destructive cholangitis (CNSDC) in the portal area in stage 1–2 PBC but was not detected in the portal area in CH–C. In stage 3–4 PBC, LTA was localized around sites of ductular proliferation at the periphery of portal tracts. IgM class anti-LTA serum titers were significantly higher in PBC than in CH–C. IgA class anti-LTA serum titers were significantly higher in PBC than in healthy subjects.

Conclusions: In the PBC livers, the profile of immunoreactivity to LTA changed markedly as the disease progressed. Sera from PBC showed higher levels of anti-LTA titers than CH–C (IgM) or from healthy subjects (IgA). The LTA-mediated immune system might affect the initiation and/or progression of PBC.