Immunophenotyping analysis in invasive micropapillary carcinoma of the breast: Role of CD24 and CD44 isoforms expression

We analyzed immunohistochemically the expression of CD24 and spliced variants of CD44v5 and v9 in invasive micropapillary carcinoma (IMPC) of the breast that is a rather aggressive tumor characterized by alteration of cells adhesion molecules, early lymph node metastases and poor prognosis. We analyzed 31 high-grade IMPCs and compared their expression to 22 high grade (G3) invasive ductal carcinomas of the breast (IDCs). We found a higher expression of CD24 in high-grade IMPCs with a peculiar inverted apical localization, compared to IDCs, showing a strong cytoplasmic staining; normal breast tissue resulted completely negative. IMPCs showed reduced expression of CD44v5 and CD44v9 compared with IDCs, but without a statistical significant difference. This study demonstrated that IMPC represents a distinct entity of breast carcinoma with high expression of CD24 with a typical inverted apical membrane pattern and reduction of CD44 isoforms v5 and v9, compared to IDCs. These features could explain the high lymph-vascular invasion propensity and higher metastatic capability of these tumors and could be a useful tool for a future targeted therapy.

Long-term results of International Breast Cancer Study Group Trial VIII: adjuvant chemotherapy plus goserelin compared with either therapy alone for premenopausal patients with node-negative breast cancer

The International Breast Cancer Study Group Trial VIII compared long-term efficacy of endocrine therapy (goserelin), chemotherapy [cyclophosphamide, methotrexate and fluorouracil (CMF)], and chemoendocrine therapy (CMF followed by goserelin) for pre/perimenopausal women with lymph-node-negative breast cancer.

Differential efficacy of three cycles of CMF followed by tamoxifen in patients with ER-positive and ER-negative tumors: long-term follow up on IBCSG Trial IX

The benefit of adjuvant chemotherapy in postmenopausal patients with estrogen receptor (ER)-positive lymph node-negative breast cancer is being reassessed.

Diffusion-weighted magnetic resonance imaging detects local recurrence after radical prostatectomy: initial experience

Current conventional cross-sectional imaging techniques, such as contrast-enhanced computed tomography and magnetic resonance imaging (MRI), are largely inaccurate in detecting local recurrence after radical prostatectomy. We report on five patients with biochemical recurrence after radical retropubic prostatectomy and pelvic lymph node dissection for whom local recurrence could only be detected with diffusion-weighted (DW) MRI. Prior to DW-MRI, all patients had negative digital rectal examinations, negative or equivocal conventional cross-sectional imaging, and negative bone scans. All suspicious lesions on DW-MRI imaging were histologically proved to be local recurrences of prostate cancer after either transrectal ultrasound-guided or transurethral biopsy. These results should encourage other centres to test our findings.

Lymphadenectomy for bladder cancer: indications and controversies

Pelvic lymph node dissection (PLND) at the time of cystectomy remains the most accurate method of staging and can have a positive impact on cancer control, and there is general agreement as to its necessity at the time of surgery. There is, however, a lack of consensus regarding the terminology of PLND and controversy concerning the optimal extent of lymph node dissection, especially because recent investigations have suggested a survival benefit with extended PLND.

Synaptic regulation of spike firing in interneurons of the striatum in vivo

The striatum, the major input nucleus of the basal ganglia, is numerically dominated by a single class of principal neurons, the GABAergic spiny projection neuron (SPN) that has been extensively studied both in vitro and in vivo. Much less is known about the sparsely distributed interneurons, principally the cholinergic interneuron (CIN) and the GABAergic fast-spiking interneuron (FSI). Here, we summarize results from two recent studies on these interneurons where we used in vivo intracellular recording techniques in urethane-anaesthetized rats (Schulz et al., J Neurosci 31[31], 2011; J Physiol, in press). Interneurons were identified by their characteristic responses to intracellular current steps and spike waveforms. Spontaneous spiking contained a high proportion (~45%) of short inter-spike intervals (ISI) of <30 ms in FSIs, but virtually none in CINs. Spiking patterns in CINs covered a broad spectrum ranging from regular tonic spiking to phasic activity despite very similar unimodal membrane potential distributions across neurons. In general, phasic spiking activity occurred in phase with the slow ECoG waves, whereas CINs exhibiting tonic regular spiking were little affected by afferent network activity. In contrast, FSIs exhibited transitions between Down and Up states very similar to SPNs. Compared to SPNs, the FSI Up state membrane potential was noisier and power spectra exhibited significantly larger power at frequencies in the gamma range (55-95 Hz). Cortical-evoked inputs had faster dynamics in FSIs than SPNs and the membrane potential preceding spontaneous spike discharge exhibited short and steep trajectories, suggesting that fast input components controlled spike output in FSIs. Intrinsic resonance mechanisms may have further enhanced the sensitivity of FSIs to fast oscillatory inputs. Induction of an activated ECoG state by local ejection of bicuculline into the superior colliculus, resulted in increased spike frequency in both interneuron classes without changing the overall distribution of ISIs. This manipulation also made CINs responsive to a light flashed into the contralateral eye. Typically, the response consisted of an excitation at short latency followed by a pause in spike firing, via an underlying depolarization-hyperpolarization membrane sequence. These results highlight the differential sensitivity of striatal interneurons to afferent synaptic signals and support a model where CINs modulate the striatal network in response to salient sensory bottom-up signals, while FSIs serve gating of top-down signals from the cortex during action selection and reward-related learning.

Appendiceal mucocele in an elderly patient: how much surgery?

Appendiceal mucoceles are rare cystic lesions with an incidence of 0.3-0.7% of all appendectomies. They are divided into four subgroups according to their histology. Even though the symptoms may vary - depending on the level of complication - from right lower quadrant pain, signs of intussusception, gastrointestinal bleeding to an acute abdomen with sepsis, most mucoceles are asymptomatic and found incidentally. We present the case of a 70-year-old patient with an incidentally found appendiceal mucocele. He was seen at the hospital for backache. The CT scan showed a vertebral fracture and a 7-cm appendiceal mass. A preoperative colonoscopy displayed several synchronous adenomas in the transverse and left colon with high-grade dysplasia. In order to lower the cancer risk of this patient, we performed a subtotal colectomy. The appendiceal mass showed no histopathological evidence of malignancy and no sign of perforation. The follow-up was therefore limited to 2 months. In this case, appendectomy would have been sufficient to treat the mucocele alone. The synchronous high-grade dysplastic adenomas were detected in the preoperative colonoscopy and determined the therapeutic approach. Generally, in the presence of positive lymph nodes, a right colectomy is the treatment of choice. In the histological presence of mucinous peritoneal carcinomatosis, cytoreductive surgery with hyperthermic intraperitoneal chemotherapy is indicated. In conclusion, mucoceles of the appendix are detected with high sensitivity by CT scan. If there is no evidence of synchronous tumor preoperatively and no peritoneal spillage, invasion or positive sentinel lymph nodes during surgery, a mucocele is adequately treated by appendectomy.

NOD2 gene variants are a risk factor for culture-positive spontaneous bacterial peritonitis and monomicrobial bacterascites in cirrhosis

Spontaneous bacterial peritonitis (SBP) is considered as result of bacterial translocation from the gastrointestinal lumen to the mesenteric lymph nodes and subsequent circulation. Variants of the NOD2 gene contribute to bacterial translocation and were associated with SBP in a recent study.

In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.

Isolation and genotyping of Toxoplasma gondii causing fatal systemic toxoplasmosis in an immunocompetent 10-year-old cat

A 10-year-old male, neutered domestic shorthair cat was presented with fever, anorexia, vomiting, and diarrhea. Serologic testing for Feline immunodeficiency virus and Feline leukemia virus were negative. Fine-needle aspirates of mesenteric lymph nodes revealed the presence of banana-shaped apicomplexan parasites. The cat died after 4 days of hospitalization. Postmortem polymerase chain reaction (PCR) analysis confirmed the presence of Toxoplasma gondii in all examined organs. Parasites were ex vivo isolated in outbred mice and subsequently transferred into cell culture. Genotyping, using genetic markers for SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico for PCR-restriction fragment length polymorphism, revealed infection with type II T. gondii displaying type II alleles at all loci except Apico, which exhibited a type I allele. This is the most frequently identified genotype among cats acting as definitive hosts in central Europe, but to the authors' knowledge, it has never been associated with systemic toxoplasmosis in an adult, immunocompetent cat.

[Juvenile sterile granulomatous dermatitis and lymphadenitis in the dog]

Juvenile sterile granulomatous dermatitis and lymphadenitis is a rare immune-mediated skin disease in young dogs. History, signalment, diagnostics, treatment, and outcome in 10 dogs are described. The age ranged from 8 - 36 weeks. The lymph nodes were enlarged in all dogs, especially the mandibular and prescapular lymph nodes. Systemic signs including fever were present in 8 dogs. Seven dogs suffered from blepharitis and painful edema of the muzzle with hemorrhagic discharge, pustules and papules. Cytology of pustules and lymph node aspirates revealed a pyogranulomatous inflammation. In 7 cases the diagnosis of juvenile sterile granulomatous dermatitis and lymphadenitis was confirmed by histology. Nine dogs were treated with prednisolone (0.5 - 1.25 mg/kg BID), H2-receptor antagonists and analgetics; all dogs were treated with antibiotics. Four dogs were treated with eye ointment containing antibiotics and glucocorticoids. The prednisolone dosage was tapered over 3 - 8 weeks. One dog had a relapse.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen

Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.

Negative regulation of NF-κB by the ING4 tumor suppressor in breast cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.