Tumor microenvironment in triple-negative breast cancer: the correlation of tumor-associated macrophages and tumor-infiltrating lymphocytes.

PURPOSE: Immune cells such as cytotoxic T cells, helper T cells, B cells or tumor-associated macrophages (TAMs) contribute to the anti-tumor response or pro-tumorigenic effect in triple negative breast cancer (TNBC). The interrelation of TAMs, T and B tumor-infiltrating lymphocytes (TILs) in TNBC has not been fully elucidated. METHODS: We evaluated the association of tumor-associated macrophages, T and B TILs in TNBC. RESULTS: TNBCs with a high CD68+, CD163+ TAMs and low CD4+, CD8+, CD20+ TILs had a significantly shorter relapse-free survival (RFS) and overall survival (OS) than those with low CD68+, CD163+ TAMs and high CD4+, CD8+, CD20+ TILs. TNBCs with high CD68+ TAMs/low CD8+ TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD68+ TAMs/high CD8+ TILs, low CD68+ TAMs/high CD8+ TILs, and low CD68+/low CD8+. TNBCs with high CD163+ TAMs/low CD8+, low CD20 + TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD163+ TAMs/high CD8+ TILs and high CD163+ TAMs /high CD20+ TILs. CONCLUSIONS: Our study suggests that TAMs further create an optimal tumor microenvironment (TME) for growth and invasion of cancer cells when evasion of immunoreactions due to T and B TILs occurs. In TNBCs, all these events combine to affect prognosis. The process of TME is highly complex in TNBCs and for an improved understanding, larger validation studies are necessary to confirm these findings.

Immunohistochemical Profile of Ameloblastic Carcinoma Arising from an Amyloid-Producing Odontogenic Tumour in a Miniature Dachshund.

A 13-year-old female miniature dachshund was presented with a centrally-located sublingual mass in the rostral mandibular region. The focally ulcerated growth completely covered the left (305) and right (405) premolar teeth and partially covered the right canine teeth (404). A punch biopsy sample revealed neoplastic proliferation of odontogenic epithelium arranged in irregular cords with frequent comedo-like necrosis. Following the initial diagnosis of ameloblastic carcinoma, a bilateral rostral hemimandibulectomy was performed. Although the detailed examination of the resected mass was consistent with the initial diagnosis, it also contained birefringent congophilic, amelogenin-labelled amyloid deposits similar to an amyloid-producing odontogenic tumour (APOT) in 30-40% of the mass, in continuity with the ameloblastic carcinoma. All neoplastic cells had diffuse moderate expression of cytokeratin (CK) AE1/AE3 and CK5, diffuse mild expression of CK14 and multifocal moderate expression of CK19. Because the APOT-like growth in the mass was histologically benign, the tumour was diagnosed as an ameloblastic carcinoma arising from an APOT.

Local extensive granulomatous inflammation of the neck region and lymphangitis caused by Lichtheimia corymbifera infection in a Japanese Black calf.

A 7-month-old female Japanese Black calf developed elongated, nodular mass measuring 30 × 16 cm extended from the retropharyngeal region to mid lateral neck region. Histological examination revealed granulomatous lymphangitis with non-septate fungal hyphae recognized throughout the lesions. Fungal culture, DNA sequencing and molecular phylogenetic tree analysis confirmed the sequence of Lichtheimia corymbifera. The lymphogenous route was speculated to be the main route of fungal spread leading to the characteristic nodular appearance of this case.

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries.

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin-4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti-Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c-erbB-2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call-Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.

Merkel Cell Carcinoma in a Steer.

Merkel cell carcinoma is a rare and aggressive cutaneous neuroendocrine tumour reported only in man, dogs and cats. A 20-month-old Japanese black fattening steer was presented with necrotic protruding skin masses over the left thoracic area and a 20 × 25 cm subcutaneous mass in the left abdominal area. Microscopical evaluation of the masses revealed cords of small to medium-sized round tumour cells with marked anisocytosis and anisokaryosis and clear and vacuolated cytoplasm, which were separated by a delicate fibrovascular stroma and arranged in a trabecular and nested pattern. Necropsy examination revealed multiple solid white nodular masses in the lungs. Immunohistochemistry (IHC) for cytokeratin (CK) 20 and CKAE1/3 revealed focal perinuclear labelling of tumour cells. IHC for the neuroendocrine markers chromogranin A and neuron specific enolase, the neuroepithelial stem cell marker nestin and the hormonal markers adrenocorticotropic hormone and calcitonin revealed diffuse cytoplasmic labelling of all tumour cells. Ultrastructurally, the tumour cells contained few neurosecretory granules and abundant glycogen pools. The tumours were diagnosed as Merkel cell carcinoma with pulmonary metastases and this case represents the first such diagnosis in cattle.

Di-(2-ethylhexyl) phthalate suppresses IL-12p40 production by GM-CSF-dependent macrophages via the PPARα/TNFAIP3/TRAF6 axis after lipopolysaccharide stimulation.

Activation of peroxisome proliferator-activated receptor α (PPARα) by di-(2-ethylhexyl) phthalate (DEHP) has an anti-inflammatory effect. This study investigated the potential combined influence of PPARα, tumor necrosis factor α-induced protein 3 (TNFAIP3/A20), and tumor necrosis factor receptor-associated factor 6 (TRAF6) on interleukin (IL)-12p40 production by macrophages exposed to DEHP and stimulated with lipopolysaccharide (LPS). LPS upregulated IL-12p40 expression by granulocyte-macrophage colony-stimulating factor-dependent macrophages (on day 9 of culture), whereas adding DEHP to cultures significantly attenuated the response of IL-12p40 to LPS stimulation. PPARα protein was also reduced by DEHP. Interestingly, transfection of macrophages with small interfering RNA (siRNA) duplexes for PPARα, TNFAIP3/A20, or dual oxidase 2 restored the response of IL-12p40 protein to LPS stimulation in the presence of DEHP. siRNAs for various protein kinase Cs (PKCs) (α, ß, γ, or δ) also restored IL-12p40 production by macrophages exposed to LPS and DEHP. While LPS upregulated both IL-12p40 and TNFAIP3/A20 production, adding DEHP to cultures dramatically reduced IL-12p40 and TNFAIP3/A20 levels. Silencing of PKCα reduced TNFAIP3/A20 production, whereas PKCγ siRNA (but not PKCß or δ siRNA) significantly increased TNFAIP3/A20. TRAF6 was also attenuated by macrophages with DEHP. The PPARα/TNFAIP3/TRAF6 axis may have an important role in the mechanism through which DEHP reduces IL-12p40 production by LPS-stimulated macrophages.

Recurrence after resection with curative intent for distal cholangiocarcinoma.

BACKGROUND: Few studies have been conducted on patterns of recurrence after resection for distal cholangiocarcinoma (DCC). The aim of this study was to investigate the incidence and pattern of recurrence after resection of DCC, and to evaluate prognostic factors for time to recurrence and recurrence-free survival (RFS). METHODS: Patients who underwent pancreatoduodenectomy with curative intent for DCC between 2001 and 2010 at one of 30 hospitals in Japan were reviewed retrospectively, with special attention to recurrence patterns. The Cox proportional hazards model was used for multivariable analysis. RESULTS: In the study interval, 389 patients underwent pancreatoduodenectomy for DCC with R0/M0 status. Recurrence developed in 213 patients (54·8 per cent). The estimated cumulative probability of recurrence was 54·3 per cent at 5 years. An initial locoregional recurrence occurred in 55 patients (14·1 per cent) and initial distant recurrence in 168 (43·2 per cent), most commonly in the liver. Isolated initial locoregional recurrence occurred in 45 patients (11·6 per cent). Independent prognostic factors for time to recurrence and RFS were perineural invasion (P = 0·001 and P = 0·009 respectively), pancreatic invasion (both P < 0·001) and lymph node metastasis (both P < 0·001). RFS worsened as the number of risk factors increased: the 5-year RFS rate was 70·6 per cent for patients without any risk factors, 50·3 per cent for patients with one factor, 31·8 per cent for those with two factors, and 13·4 per cent when three factors were present. CONCLUSION: More than half of patients with DCC experienced recurrence after R0 resection, usually within 5 years. Perineural invasion, pancreatic invasion and positive nodal involvement are risk factors for recurrence.

Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens).

A 9-year-old male red panda (Ailurus fulgens) became emaciated and died. Necropsy examination revealed systemic lymphadenomegaly. The liver, lungs and left kidney contained multifocal yellow nodules. Microscopical examination revealed granulomatous inflammation in the liver, lungs, kidney, spleen and lymph nodes, with numerous acid-fast bacilli. Sequencing of genetic material isolated from the tissues classified the pathogen as Mycobacterium gastri. Lymphoma was found in the liver, lungs, kidney and lymph nodes. The neoplastic cells were strongly labelled for expression of CD3, Ki67 and proliferating cell nuclear antigen by immunohistochemistry. This is the first report of M. gastri infection with T-cell lymphoma in a red panda.

The hypusine cascade promotes cancer progression and metastasis through the regulation of RhoA in squamous cell carcinoma.

Metastasis is a critical factor contributing to poor prognosis in cancer, but the underlying mechanisms of metastasis are still poorly understood. We established a highly metastatic cell subline (HOC313-LM) derived from an oral squamous cell carcinoma cell line (HOC313) for uncovering the mechanisms of metastasis, and identified deoxyhypusine synthase (DHPS) as a metastasis-associated gene within the specific amplification at 19p13.2-p13.13 in HOC313-LM. DHPS-mediated hypusine-modification of eukaryotic translation factor 5A facilitated the translation of RhoA, resulting in the activation of the RhoA signaling pathway and leading to not only increased cell motility, invasion and metastasis of cancer cells in vitro, but also increased tumor growth in vivo. Moreover, the use of N1-Guanyl-1,7-diaminoheptane, a DHPS inhibitor, resulted in a significant decrease in tumor formation in vivo. In patients with esophageal squamous cell carcinoma (ESCC), overexpression of DHPS in ESCC tumors was significantly associated with worse recurrence-free survival, and correlated with distant metastasis. The elucidation of these molecular mechanisms within the hypusine cascade suggests opportunities for novel therapeutic targets in SCC.

Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients.

Allogeneic hematopoietic stem cell transplantation (HSCT) is one of curative treatment options for patients with hematologic malignancies. Although GVHD mediated by the donor's T lymphocytes remains the most challenging toxicity of allo-HSCT, graft-versus-leukemia (GVL) effect targeting leukemic cells, has an important role in affecting the overall outcome of patients with AML. Here we comprehensively characterized the TCR repertoire in patients who underwent matched donor or haplo-cord HSCT using next-generation sequencing approach. Our study defines the functional kinetics of each TCRA and TCRB clone, and changes in T-cell diversity (with identification of CDR3 sequences) and the extent of clonal expansion of certain T-cells. Using this approach, our study demonstrates that higher percentage of cord-blood cells at 30 days after transplant was correlated with higher diversity of TCR repertoire, implicating the role of cord-chimerism in enhancing immune recovery. Importantly, we found that GVHD and relapse, exclusive of each other, were correlated with lower TCR repertoire diversity and expansion of certain T-cell clones. Our results highlight novel insights into the balance between GVHD and GVL effect, suggesting that higher diversity early after transplant possibly implies lower risks of both GVHD and relapse following the HSCT transplantation.

Modulating effect of low level-laser therapy on fibrosis in the repair process of the tibialis anterior muscle in rats.

The treatment of muscle injuries is a common practice at rehabilitation centers. Low-level laser therapy (LLLT) has demonstrated positive effects regarding the modulation of the inflammatory response, the enhancement of the tissue repair process and the prevention of fibrosis. The aim of the present study was to evaluate the effects of LLLT on morphological aspects of muscle tissue, collagen remodeling and activity of matrix metalloproteinase 2 (MMP-2) in rat skeletal muscle following acute injury. Wistar rats were divided into five groups: (1) control group (n = 10), (2) sham group (n = 10), (3) LLLT group (n = 30), (4) non-treated injury group (n = 30) and (5) injury + LLLT group (n = 30). Cryoinjury was performed on the belly of the tibialis anterior (TA) muscle. LLLT was performed daily with an AlGaAs laser (780 nm; beam spot of 0.04 cm(2), output power of 40 mW, power density of 1 W/cm(2), energy density of 10 J/cm(2) and 10-s exposure time). Animals were euthanized at 1, 3 and 7 days. The TA muscles were removed and weighed. Morphological aspects were evaluated using H & E staining. The amount and distribution of collagen fibers were evaluated by picrosirius staining. Characterization and activity of MMP-2 were evaluated by zymography and Western blot techniques, respectively. The results revealed that LLLT induced a reduction in inflammatory infiltrate and myonecrosis after 1 day, an increase in the number of blood vessels after 3 and 7 days as well as an increase in the number of immature muscle fibers and MMP-2 gelatinase activity after 7 days. In conclusion, LLLT has a positive effect on the inflammatory process, MMP2 activity and collagen organization and distribution in the repair process of rat skeletal muscle.

Adhesion of Streptococci to various orthodontic composite resins.

BACKGROUND: This investigation aimed to determine quantitatively the adhesion of Streptococcus mutans and Streptococcus sobrinus to orthodontic composite resins that were tested simultaneously using radio-markers. METHODS: Seven orthodontic composite resins were classified into seven groups: BeautyOrtho Bond (GI), Blugloo (GII), Enlight (GIII), Grengloo (GIV), Kurasper F (GV), Transbond CC (GVI) and Turbo Bond II (GVII). Thirty 4 x 4 x 1 mm blocks of each orthodontic composite resin were made (a total of 210 blocks). Both Streptococcus species were cultivated independently. For the quantitative analysis, radioactive markers were used to codify the bacteria ((3) H for Streptococcus mutans and (14) C for Streptococcus sobrinus). The blocks were submerged in a solution with microorganisms previously radiolabelled for 2 hours at 37 °C in constant movement. The blocks were placed in a combustion system to quantify the Streptococcus adhering to the surface of the materials by capturing the residues and measuring the radiation. RESULTS: Significant differences in bacterial adhesion were found among the groups. The lowest significant scores for both microorganisms were observed in GIII. CONCLUSIONS: The orthodontic composite resin evaluated in GIII exhibited the lowest adhesion of Streptococcus mutans and Streptococcus sobrinus, which may reduce enamel demineralization and the risk of white spot lesion formation.

Clinical and safety profiles of bipolar transurethral vaporization of the prostate in saline: a preliminary report.

Transurethral vaporization of the prostate in saline (TURisV) is an innovative endoscopic surgical modality for the treatment of benign prostatic hyperplasia (BPH) that vaporizes prostate tissue using a uniquely designed mushroom electrode. TURisV promises instant hemostatic tissue ablation under saline irrigation and offers clinical advantages for endoscopic BPH operations. From July 2008 to February 2009, TURisV was performed in 17 cases with clinically significant BPH. Median operation time was 127.0 min and median volume of vaporized prostate tissue was 41.1 g. Median International Prostate Symptom Score improved from 20 to 4 after 12 months. Median maximum flow rate increased from 5.3 mL/s to 13.8 mL/s after 12 months. Postoperative median residual urine improved from 48.0 mL to 7.0 mL after 12 months. No changes in hemoglobin or electrolyte levels were seen postoperatively. Our results suggest that TURisV is a safe and efficacious treatment for BPH.

Morphological analysis of collapsed regions in osteonecrosis of the femoral head.

In order to investigate the mechanisms of collapse in osteonecrosis of the femoral head, we examined which part of the femoral head was the key point of a collapse and whether a collapsed region was associated with the size of the necrotic lesion. Using 30 consecutive surgically removed femoral heads we retrospectively analysed whole serial cut sections, specimen photographs, specimen radiographs and histological sections. In all of the femoral heads, collapse consistently involved a fracture at the lateral boundary of the necrotic lesion. Histologically, the fractures occurred at the junction between the thickened trabeculae of the reparative zone and the necrotic bone trabeculae. When the medial boundary of the necrotic lesion was located lateral to the fovea of the femoral head, 18 of 19 femoral heads collapsed in the subchondral region. By contrast, when the medial boundary was located medial to the fovea, collapse in the subchondral region was observed in four of 11 femoral heads (p = 0.0011). We found that collapse began at the lateral boundary of the necrotic lesion and that the size of the necrotic lesion seemed to contribute to its distribution.

Altered gene expressions of ghrelin, PYY, and CCK in the gastrointestinal tract of the hyperphagic intrauterine growth restriction rat offspring.

Intrauterine growth restriction (IUGR) is associated with a substantially greater incidence of metabolic syndrome in adulthood. Animal studies have shown that IUGR offspring are hyperphagic during the early postnatal period and therefore exhibit obesity. The molecular mechanisms underlying food intake regulation in the gastrointestinal tract have not been clarified in IUGR. In the present study, we utilized a rat model of IUGR by restricting the food intake of the mother (50% of the normal intake, ad libitum; FR group) from day 7 of gestation until delivery. Pups from undernourished mothers were fostered by control mothers. We examined the food intake and assessed the gene expressions of ghrelin, peptide YY (PYY), and cholecystokinin (CCK) in the alimentary tract of male newborns (postnatal day1) and adult offspring (age, 7 months). Compared to the offspring whose mothers received the standard diet ad libitum (CON offspring), FR offspring were hyperphagic from the weaning time until the end of the experiment, and resulted in a heavier final weight. Both newborn and adult FR offspring had higher ghrelin gene expression in the stomach and higher ghrelin plasma levels than did the controls. Although the gastrointestinal gene expressions and plasma levels of the anorexic peptides, PYY and CCK, were elevated in the FR newborns, they decreased in the FR adults. Our findings suggest that the altered gene expressions of orexigenic and anorexigenic gut peptides in the gastrointestinal tract in the maternal undernutrition-induced IUGR offspring provide a potential mechanism to explain hyperphagia and obesity seen in these offspring.

Assessment of hemorrhage in pituitary macroadenoma by T2*-weighted gradient-echo MR imaging.

BACKGROUND AND PURPOSE: Intratumoral hemorrhage occurs frequently in pituitary macroadenoma and manifests as pituitary apoplexy and recent or old silent hemorrhage. T2*-weighted gradient-echo (GE) MR imaging is the most sensitive sequence for the detection of acute and old intracranial hemorrhage. T2*-weighted GE MR imaging was used to investigate intratumoral hemorrhage in pituitary macroadenomas. MATERIALS AND METHODS: Twenty-five consecutive patients who underwent total or subtotal resection of pituitary macroadenoma with heights from 17 to 53 mm, including 1 patient with classic pituitary apoplexy, underwent MR imaging before surgery, including T2*-weighted GE MR imaging. For histologic assessment of the hemorrhage in whole surgical specimens, we used hematoxylin-eosin staining. RESULTS: T2*-weighted GE MR imaging detected various types of dark lesions, such as "rim," "mass," "spot," and "diffuse" and combinations, indicating clinical and subclinical intratumoral hemorrhage in 12 of the 25 patients. The presence of intratumoral dark lesions on T2*-weighted GE MR imaging correlated significantly with the hemorrhagic findings on T1- and T2-weighted MR imaging (P < .02 and <.01, respectively), and the surgical and histologic hemorrhagic findings (P < .001 and <.001, respectively). CONCLUSION: T2*-weighted GE MR imaging could detect intratumoral hemorrhage in pituitary adenomas as various dark appearances. Therefore, this technique might be useful for the assessment of recent and old intratumoral hemorrhagic events in patients with pituitary macroadenomas.

Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity.

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.