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Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E-cadherin loss. Focal activation of P-cadherin expression in tumor cells that are deficient for E-cadherin occurs in a subset of ILCs. Switching from an E-cadherin deficient to P-cadherin proficient status (EPS) partially restores cell-cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52-year-old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E-cadherin and P-cadherin. CDH1/E-cadherin mutations were determined by next-generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1/E-cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E-cadherin-negative and P-cadherin-negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter-cryptal ILC cells switched to a P-cadherin-positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment-induced EPS and conversion to cohesive growth., (© 2024 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

3D printed clamps provide multiple advantages compared to potting for the fixation of spinal specimens and in a recent study, superior fixation stability was reported. The aim of this study was to evaluate the fixation efficacy of 3D printed vertebra clamps during routine application and to present and evaluate a novel clamp for sacrum fixation. Further, public access to the template files is provided. 98 human single-level cadaveric specimens were biomechanically tested in flexion-extension (FE), lateral bending (LB), axial rotation (AR), anteroposterior shear (AS), lateral shear (LS) and axial compression-decompression (AC). Loading amplitudes were +/-7.5 Nm for FE, LB and AR, +/- 150 N for AS and LS and + 400/-100 N for AC. The novel sacrum clamp was used in 8 specimens. The median relative motion between clamps and specimens was 0.6° in FE, 0.7° in LB, 0.3° in AR, 0.5 mm in AS, 0.5 mm in LS and 0.1 mm in AC. With sacrum clamps, the median relative motion was 0.3° in FE, 0.1° in LB, 0.08° in AR, 0.8 mm in AS, 0.7 mm in LS and 0.2 mm in AC. The vertebra clamps used during routine testing provided better stability compared to the values in the literature in all six loading directions (p < 0.05). The sacrum clamp showed superior anchoring stability in three loading directions compared to the caudal vertebra clamps (p < 0.05), while inferior stability was measured in AS (p < 0.001). We conclude that 3D printed vertebra clamps and 3D printed sacrum clamps represent reliable methods for specimen fixation during routine biomechanical testing., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The genetic hallmark of epithelioid hemangioma (EH) is the presence of recurrent gene fusions involving FOS and FOSB transcription factors, which occur in one-third of the cases. Certain clinical, pathologic, and genotypic correlations have been described, with FOS-related fusions being more often detected in skeletal and cellular variants of EH, while FOSB gene rearrangements are more commonly associated with atypical histologic features and penile location. These fusions are infrequently detected in the cutaneous or head and neck EH. Overall, two-thirds of EH lack these canonical fusions and remain difficult to classify, especially when associated with atypical features and/or clinical presentations. Triggered by an index case of an intravascular soft tissue EH with a novel GATA6-FOXO1 gene fusion by targeted RNA sequencing (Archer® FusionPlex® Sarcoma Panel), we have investigated 27 additional EH cases negative for FOS and FOSB gene rearrangements for this novel abnormality to determine its recurrent potential, and its association with clinical and pathologic features. Four additional EH cases were found to display GATA6-FOXO1 fusions (18%). There were three females and two males, with a mean age of 32 years old. Three lesions occurred in the head and neck (dura, nasopharyngeal, and cheek), one in the back and one in the leg. Two of these lesions were cutaneous and one was intravascular in the subcutis of the leg. Microscopically, the tumors showed a variegated morphology, with alternating vasoformative and solid components, extravasated red blood cells and mild to moderate cytologic atypia. None showed brisk mitotic activity or necrosis. Tumors were negative for FOS and FOSB by immunohistochemistry. In conclusion, we report a new GATA6-FOXO1 fusion in a subset of EH, with a predilection for skin, and head and neck location. The relationship of this novel molecular subset with the more common FOS/FOSB fusion-positive EH remains to be determined.

Background: Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 (TREX2) locus in laryngeal (n = 256) and colorectal cancer cases (n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA)., Results: Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts (p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 (p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients (p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression., Conclusions: The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies.

Purpose: To evaluate whether 3D-printed vertebrae offer realistic haptic simulation of posterior pedicle screw placement and decompression surgery with normal to osteoporotic-like properties., Methods: A parameterizable vertebra model was developed, adjustable in cortical and cancellous bone thicknesses. Based on this model, five different L3 vertebra types (α, β, γ1, γ2, and γ3) were designed and fourfold 3D-printed. Four spine surgeons assessed each vertebra type and a purchasable L3 Sawbones vertebra. Haptic behavior of six common steps in posterior spine surgery was rated from 1 to 10: 1-2: too soft, 3-4: osteoporotic, 5-6: normal, 7-8: hard, and 9-10: too hard. Torques were measured during pedicle screw insertion., Results: In total, 24 vertebrae (six vertebra types times four examiners) were evaluated. Mean surgical assessment scores were: α 3.2 ± 0.9 (osteoporotic), β 1.9 ± 0.7 (too soft), γ1 4.7 ± 0.9 (osteoporotic-normal), γ2 6.3 ± 1.1 (normal), and γ3 7.5 ± 1.1 (hard). All surgeons considered the 3D-printed vertebrae α, γ1, and γ2 as more realistic than Sawbones vertebrae, which were rated with a mean score of 4.1 ± 1.7 (osteoporotic-normal). Mean pedicle screw insertion torques (Ncm) were: α 32 ± 4, β 12 ± 3, γ1 74 ± 4, γ2 129 ± 13, γ3 196 ± 34 and Sawbones 90 ± 11., Conclusions: In this pilot study, 3D-printed vertebrae displayed haptically and biomechanically realistic simulation of posterior spinal procedures and outperformed Sawbones. This approach enables surgical training on bone density-specific vertebrae and provides an outlook toward future preoperative simulation on patient-specific spine replicas. These slides can be retrieved under Electronic Supplementary Material.

A 50 year old woman underwent laparoscopic supracervical hysterectomy because of symptomatic fibroids. Histologic examination of samples obtained after morcellation revealed typical uterine leiomyomas in all samples investigated. 28 and 47 months later, respectively, the patient presented with peritoneal spreading of nodules that were surgically removed and histologically classified as leiomyosarcoma. In 3/4 of samples obtained after morcellation copy number/SNP-array hybridization showed complex genomic alterations widely identical to the pattern characterizing the sarcoma. Therefore, we conclude that the leiomyosarcoma had unambiguously developed from one of the leiomyomas as a result of secondary genetic alterations i.e. a rearrangement of ALK and a del(14q). The case is challenging the current risk estimates for spreading of unexpected malignant uterine tumors due to power morcellation and highlights the relevance of certain genetic alterations for rare malignant transformation of uterine benign smooth muscle tumors., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interests.

Nondestructive, sublethal, and sensitive health monitoring tools are needed to assess the health of freshwater mussels (family Unionidae). Recent developments to standardize hemocyte characterization have assisted in the hematologic assessment of wild and captive freshwater mussels. In this study, preliminary baseline hematological reference ranges were established for wild mapleleaf mussels Quadrula quadrula (n = 14) and threeridge mussels Amblema plicata (n = 20) collected from the Muskingum River in Devola, Ohio. Mussels were collected from the wild, and hemolymph was sampled from each mussel in the field upon capture (baseline sample). They were then transported live to a propagation facility. Subsequent hemolymph samples were collected at 2 and 4 weeks and quarterly thereafter for 11 months following translocation. Hemocyte counts, hemocyte morphology, and hemolymph chemistry (Na + , Cl - , Mg 2+ , P 3- , K + , Ca 2+ , glucose, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase]) were measured from each sample on each sampling occasion. Hemocyte counts were consistently greater in Q. quadrula than in A. plicata following transfer to captivity. Baseline hemocyte morphology and hemolymph chemistry varied between species. This study provides a foundation of reference ranges for hemocyte characterization for Q. quadrula, and A. plicata and a preliminary understanding of how hemocyte character might be expected to change when wild mussels are translocated into captivity, and thus be a useful technique for monitoring the health of freshwater mussels., (© 2018 American Fisheries Society.)

Background: Placement of the glenoid baseplate is of paramount importance for the outcome of anatomical and reverse total shoulder arthroplasty. However, the database around glenoid size is poor, particularly regarding small scapulae, for example, in women and smaller individuals, and is derived from different methodological approaches. In this multimodality cadaver study, we systematically examined the glenoid using morphological and 3D-CT measurements., Methods: Measurements of the glenoid and drill hole tunnel length for superior baseplate screw placement were recorded to define size of the glenoid and the distance to the scapular notch on cadaveric specimens. Glenoid angles were determined on both, 3D-CT-scans of the thoraxes using the Friedman method and on subsequently isolated scapulae from 18 male and female donors (average 84 years, range 60-98 years)., Results: Mean glenoid height was 36.6 mm ± 3.6, and width 27.8 mm ± 3.1 with a significant sex dimorphism (p ≤ 0.001): in males, glenoid height 39.5 mm ± 3.5, and width 30.3 mm ± 3.3, and in females, glenoid height 34.8 mm ± 2.2, and width 26.2 mm ± 1.6. The average distance from the superior screw entry to its exit in the scapular notch measured by calliper was 27.2 mm ± 6.0 with a sex difference: in males, 29.4 mm ± 5.7, and in females, 25.8 mm ± 5.9 mm with a minimum recorded distance of 15 mm. Measured by CT, the mean inclination angle for male and female donors combined was 13.0° ± 7.0, and the ante-/retroversion angle -1.0° ± 4.0°., Conclusion: This study is one of the first to combine dissection, including drill holes, with anatomical measurements and radiological data. In some women and smaller individuals, smaller baseplates should be selected. The published safe zone of 20 mm is generally feasible for superior screw placement, however, in small patients this distance may be substantially shorter than expected and start as of 13 and 15 mm, respectively. No correlation between glenoid height or width with the length of our drilling canal towards the scapular notch was found. Preoperative CT-based treatment planning to determine version and inclination angles is recommended.

Deleted in malignant brain tumor 1 (DMBT1) is involved in innate immunity and epithelial differentiation. Previous studies in adults indicated a strong intestinal expression of DMBT1 and an important role in inflammatory bowel diseases. Here, we analyzed the DMBT1 expression in the fetal gastrointestinal system depending on gestational age and in patients with necrotizing enterocolitis (NEC), volvulus, intestinal perforation (IP), or herniation, representing typical diseases of preterm and term infants. We used immunohistochemistry and RNA in situ hybridization to detect DMBT1 protein and mRNA in fetal tissues, supplemented by postmortem analysis of DMBT1 expression in died newborns and analysis of surgically removed tissues. DMBT1 expression is detectable in the early developmental stages of the gastrointestinal system. In NEC, volvulus, IP, or herniation, characterized by high systemic inflammatory responses, DMBT1 expression is strongly increased. High DMBT1 expression was also found in the bile ducts of older infants with sepsis or cholestasis. The study shows that DMBT1 expression is observed in the developing gastrointestinal system and up-regulated in infants with NEC, volvulus, IP, and herniation. DMBT1 may play a role in epithelial differentiation and local innate immunity during neonatal inflammatory bowel processes.

Uterine fibroids rank among the most frequent symptomatic human tumors at all. Recent data suggest that mutations of the mediator subcomplex 12 gene (MED12) and rearrangements of the gene-encoding high-mobility group protein AT-hook 2 (HMGA2) characterize major genetic subtypes of these tumors, which, for example, differ by their average size. Herein, we have investigated a total of 289 fibroids from 120 patients. Of these fibroids, 256 were fully genetically analyzed. Of the latter group, 20 (7.8%) fibroids had a chromosomal rearrangement of 12q14-15 reflecting a rearranged allele of HMGA2 and 179 (69.9%) fibroids had a mutation of MED12. The remaining tumors had either another genetic abnormality or no detectable abnormality at all. We were able to demonstrate that tumors of both groups also display striking differences of their frequency in individual patients. Whereas 70.0% (14/20) HMGA2-mutated fibroids made their appearance as solitary nodules, 85.5% (153/179) MED12-mutated fibroids occurred as multiple nodules as a rule of independent clonal origin, as reflected by different MED12 mutations. These findings are likely to point to a different pathogenesis of both types of fibroids. In the predominant of these groups so far, an unknown "mutator" may cause independent mutations of MED12, resulting in an independent clonal outgrowth of nodules. Furthermore, the low but existing risk of MED12-mutated fibroids to undergo malignant transformation after a leiomyoma-STUMP (smooth muscle tumors of uncertain malignant potential)-leiomyosarcoma sequence excludes the latter mutation as a suitable stand-alone marker for benign growth.

Purpose: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid-induced differentiation to target the stem-like tumor cell pool could have therapeutic implications., Experimental Design: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid-containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model., Results: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo., Conclusions: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma., (Copyright (c) 2010 AACR.)

Objective: Bacterial endocarditis is a frequent infectious cardiac disease, especially in patients with congenital or acquired heart defects. It is characterized by bacterial colonization of the heart valves and the appearance of vegetations consisting of fibrin, blood cells, and bacteria. The glycoprotein Deleted in Malignant Brain Tumors 1 is a scavenger receptor cysteine-rich protein with functions in innate immunity and epithelial differentiation. Because of the aggregating capacity of Deleted in Malignant Brain Tumors 1, we hypothesized that an up-regulation in bacterial endocarditis may be linked to the development of vegetations., Methods: Heart tissue of 19 patients with bacterial endocarditis and 10 controls without bacterial endocarditis was analyzed by immunohistochemistry. The effect of human recombinant Deleted in Malignant Brain Tumors 1 on erythrocyte aggregation was measured using an automated red blood cell aggregometer MA1. Binding of human recombinant Deleted in Malignant Brain Tumors 1 to erythrocyte membranes, platelets, fibrin, and fibrinogen was analyzed by Western blotting and enzyme-linked immunosorbent assay., Results: Deleted in Malignant Brain Tumors 1 expression was up-regulated in affected heart valves with bacterial endocarditis and limited to the colonizing bacteria on the heart valves and granulocyte-depleted fibrin/fibrinogen formations, and around localized atheromatosis. Patients with aggressive bacteria showed higher DMBT1 levels than patients with less aggressive bacteria. Human recombinant Deleted in Malignant Brain Tumors 1 aggregates erythrocytes and binds to erythrocyte membranes, platelets, and fibrin/fibrinogen., Conclusion: Deleted in Malignant Brain Tumors 1 up-regulation at sites of bacterial endocarditis, its association with platelets and fibrin/fibrinogen, and its ability to aggregate erythrocytes through binding to their membranes indicate a potential role in the development of vegetations and thrombosis.

Head and neck squamous cell carcinoma has still a poor prognosis. Since angiogenesis is crucial for tumor growth, a better understanding of the potential clinical relevance as well as the interactions between the numerous proangiogenic growth factors is essential to develop improved therapeutic strategies in these tumors. Expression levels of eight growth factors known to induce angiogenesis (HGF, bFGF, VEGF-A, VEGF-D, PDGF-AB, PDGF-BB, G-CSF, and GM-CSF) were quantitatively measured by ELISA in homogenates of 41 head and neck squamous cell carcinomas. In addition, microvessel density and protein localization of growth factors were assessed by immunohistochemistry. Statistical analysis was performed to assess interrelationships between growth factors analyzed and to correlate protein levels with patient outcome. In 90% of the tissues at least 4/8 growth factors analyzed were detectable. Highest amounts and most frequent expression were found for HGF, bFGF and VEGF-A while PDGF-AB and PDGF-BB were present in two-thirds and G-CSF and GM-CSF in approximately half of the cases. Although there was no significant relation to microvessel density, we identified significant associations for bFGF with HGF and G-CSF as well as of PDGF-AB with those of VEGF-A and PDGF-BB. For the first time we demonstrate that expression levels of HGF as well as that of bFGF and G-CSF in head and neck squamous tumors are negative prognostic factors for patient survival. Our data indicate a network of interrelated and prognostically relevant growth factors in these tumors that have to be taken into consideration when planning an antiangiogenic and antitumor therapy.

LPSs getting access to the circulation of mammalian organisms cause typical systemic inflammatory reactions with symptoms characteristic for acute sepsis. One possibility to attenuate LPS effects is to expose a host to a challenge with low LPS doses, which results in the establishment of "endotoxin tolerance" (ET). Because the microcirculation is of particular importance in LPS action, it seemed of interest to analyze leukocyte-endothelial interactions in the mesentery and liver once endotoxin tolerance has been established and are challenged with LPS. The mesenteric and hepatic microcirculation was investigated by intravital microscopy. After induction of ET LPS, shock was induced by i.v. injection of LPS, and microcirculation of the mesentery and liver was examined. Endotoxin tolerance resulted in reduced ex vivo TNF-alpha synthesis of whole blood. In vivo LPS caused no increase of body temperature. In sinusoids, LPS challenge increased adherence of leukocytes in naive rats, which was almost completely prevented by ET induction. In contrast, in postsinusoidal venules, leukocyte adherence was more intense after ET induction and subsequent to LPS application. Similarly, in postcapillary mesenteric venules, increased adherence of leukocytes after LPS challenge in the ET group was observed. After LPS injection, the endothelial barrier was more disturbed in the nontolerant group when compared with the ET group. Soluble L-selectin and intercellular adhesion molecule were elevated in both ET and untreated rats. Endotoxin tolerance influences leukocyte-endothelial interaction differentially depending on organ and vessel area.

A gluten-free diet (GFD) is the sole effective long-lasting treatment of celiac disease. Four monoclonal antibodies (Abs) were prepared by immunization of animals kept on GFD with gliadin. The specificity of these Abs to decapeptides of alpha- and gamma-gliadin and omega-secalin was analyzed by the PEPSCAN technique. Repetitive sequences of alpha- and gamma-gliadin and omega-secalin containing the motifs QPFPXQ (X = Q, L, P) were recognized by all Abs tested. These Abs also frequently reacted with peptides containing the sequences QQSFPQQ, QQTFPQP, and QPFRPQ. On the basis of PEPSCAN results two Abs--8D4 and 7C6--were selected for the construction of a new ELISA kit for the detection of gliadin in food. The comparison of data obtained using the newly developed ELISA kit and commercially available ones indicated that Abs selection on the basis of their fine specificity to gliadin is useful for sensitive detection of gliadin in foods.

Critical assessment of intestinal immune responses requires the ability to characterize leukocytes from different anatomic locations as leukocytes from inductive sites such as Peyer's patches and lymphoid follicles vary significantly from their effector counterparts, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). This study describes (1) methods developed to isolate specific intestinal leukocyte populations with high yield and purity, (2) difficulties encountered in establishing a panel of monoclonal antibodies to assess phenotype, and (3) the phenotypic characterization of effector and inductive sites in the feline small intestine. We found that the phenotypic distribution of feline intestinal leukocytes was similar to that found in other species such as humans, macaques and mice. The majority of IEL were CD5(+) T-cells with less than 7% B-cells. CD8(+) T-cells comprised approximately 60% of the IEL with roughly half displaying CD8alphaalpha homodimers. Approximately 10% of IEL were CD4(+) T-cells. In the LPL, CD4(+) T-cells predominated at 42%, with 33% CD8(+) T-cells and 10% B-cells. As would be expected, B-cells predominated in Peyer's patches with 40% B-cells, 28% CD4(+) T-cells and 20% CD8(+) T-cells. Increased MHCII expression was found in the Peyer's patches as compared to the IEL and LPL. B7.1 expression was significantly higher in mucosal leukocyte populations as compared to organized lymphoid tissue in the periphery with expression detected on 65% of IEL and 53% of LPL. Plasma cells were found in all regions of small intestine examined with greater numbers in lamina propria and Peyer's patches. Lymphoblasts were only identified in inductive tissue. In general, no differences were found between the phenotype of mucosal leukocyte populations from specific pathogen free or random source cats. However, the percentage of CD4(+) CD25(+) T-cells was significantly greater in both IEL and LPL from random source animals. This study provides techniques and a baseline from which future studies of the feline intestinal immune system can be conducted.

Listeria monocytogenes is an attractive biologic vaccine vector against HIV because it induces a strong cell mediated immune response, can be delivered by mucosal routes, can be readily manipulated to express viral antigens, and is easy and inexpensive to produce. Proof of concept studies have been performed using HIV Gag expressing recombinant L. monocytogenes in the mouse. Here we report the development and validation of recombinant L. monocytogenes to be evaluated in the FIV/cat model of HIV. Using a simplified approach to introduce individual and polyprotein FIV gag genes, we show that recombinant L. monocytogenes containing the entire gag expresses the full-length Gag polyprotein in a soluble secreted form. A DNA vaccine plasmid (pND14-Lc-env) that replicates in Gram positive bacteria and contains the FIV SU (gp100) and the ectodomain of TM (gp40) in a eukaryotic expression cassette was transfected into LM-gag to create LM-gag/pND14-Lc-env. After infection of target cells with LM-gag/pND14-Lc-env in vitro, both FIV Gag and Env proteins were detected in soluble cell lysates. Whether previous exposure to L. monocytogenes affects the immunogenicity of LM-gag/pND14-Lc-env was determined in cats infected with wild-type L. monocytogenes orally and/or subcutaneously. After a single oral dose of LM-gag/pND14-Lc-env, cats with existing anti-L. monocytogenes immune responses developed anti-FIV Gag IgA titers in vaginal secretions, saliva, and feces. Similarly, FIV Gag and Env specific IFN-gamma ELISPOT responses were measurable in spleen and lymph node but at a statistically higher frequency in cats exposed to a single subcutaneous dose of wild-type L. monocytogenes versus cats exposed both subcutaneously and orally. The FIV/cat model will provide a useful challenge system to determine whether recombinant L. monocytogenes can protect against a lentivirus in its natural host after challenge by the routes common to HIV transmission.

Background & Aims: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS , an additional component of the MAPK-signalling pathway., Methods: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced., Results: We detected BRAF mutations in 2 of 19 cases and NRAS mutations in none of the cases. Mutations in exon 15 of BRAF were present in only 1 tumor (1 of 19 cases). The A1800T base exchange represented a novel mutation and resulted in a K600N transition in an AM from a 96-year-old white man who presented with rectal bleeding and painful sitting of a few weeks' duration. The second positive AM case, a 69-year-old white man who presented with painless rectal bleeding and clinical symptoms of an intestinal constipation showed a novel missense mutation (C1327T leading to R443W conversion) in BRAF exon 11. None of the AM cases displayed the oncogenic V599E mutation preponderating in CM., Conclusions: With regard to the frequency of V599E BRAF mutations, AM significantly differs from CM (P < or = .0001), which suggests that BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.

Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild-type allele. Inactivation of APC leads to impaired degradation of beta-catenin, thereby promoting continuous cell-cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ-line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second-hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of beta-catenin to the mutational status of the APC gene. Exon 3 of the beta-catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated beta-catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of beta-catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP-associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients., (Copyright 2004 Wiley-Liss, Inc.)

Recombinant Listeria monocytogenes has many attractive characteristics as a vaccine vector against human immunodeficiency virus (HIV). Wild-type and attenuated Listeria strains expressing HIV Gag have been shown to induce long-lived mucosal and systemic T-cell responses in mice. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated recombinant L. monocytogenes in a challenge system. Five cats were immunized with recombinant L. monocytogenes that expresses the FIV Gag and delivers an FIV Env-expressing DNA vaccine (LMgag/pND14-Lc-env). Control cats were either sham immunized or immunized with wild-type L. monocytogenes (LM-wt). At 1 year after vaginal challenge, provirus could not be detected in any of the nine tissues evaluated from cats immunized with the recombinant bacteria but was detected in at least one tissue in 8 of 10 control animals. Virus was isolated from bone marrow of four of five LMgag/pND14-Lc-env-immunized cats by use of a stringent coculture system but required CD8(+) T-cell depletion, indicating CD8(+) T-cell suppression of virus replication. Control animals had an inverted CD4:CD8 ratio in mesenteric lymph node and were depleted of both CD4(+) and CD8(+) intestinal epithelial T cells, while LMgag/pND14-Lc-env-immunized animals showed no such abnormalities. Vaginal FIV-specific immunoglobulin A was present at high titer in three LMgag/pND14-Lc-env-immunized cats before challenge and in all five at 1 year postchallenge. This study demonstrates that recombinant L. monocytogenes conferred some control of viral load after vaginal challenge with FIV.

Primary cutaneous melanomas and melanoma metastases were examined for differential gene expression using subtractive suppression hybridization in a search for any genes associated with metastasis. Generating a subtracted library of candidate genes up-regulated in melanoma metastases, this library contained 8 different cDNAs, among them a cDNA fragment of the syntenin gene which was overexpressed in further independent melanoma resection specimens on cDNA Southern blots when compared to acquired melanocytic nevi from which melanomas are known to arise. Upon immunohistochemistry, the syntenin protein expression was detected in the cytoplasm of primary cutaneous melanomas and melanoma metastases. Melanoma metastases exhibited higher proportions of tumour cells positive for syntenin immunostaining in comparison to acquired melanocytic nevi or non-metastasizing primary melanomas which was statistically significant. In addition to melanoma, gastric cancer tissues exhibited a higher syntenin mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Altogether, we here first describe the detection of the syntenin protein in resection specimens of melanocytic lesions. We conclude that melanoma metastasis is associated with increased expression of the syntenin gene which may participate in signal transduction and cell adhesion via the multifunctional protein-binding properties of its tandem PDZ domains.

Seeking to identify melanoma-associated genes by comparing gene expression in uncultured primary melanoma specimens with those in nevi, from which melanomas often are known to arise, we applied subtractive suppression hybridization. Generating a subtracted library of candidate genes up-regulated in primary melanomas, this library contained cDNA fragments of the genes encoding heat shock cognate protein (HSP73) and major histocompatibility complex (HLA-DR) which were overexpressed in further 19 independent melanoma resection specimens on cDNA Southern blots when compared to 19 acquired melanocytic nevi. Upon immunohistochemistry, HSP73 protein expression was detected in the cytoplasm of melanoma cells in primary tumours and metastases. In primary melanomas, the proportion of HSP73 protein expressing cells correlated with tumour thickness according to Breslow which was statistically significant. HSP73 immunostaining was stronger in melanoma metastases when compared with acquired melanocytic nevi which was statistically significant. In addition to melanoma, gastric and uterus cancer tissues exhibited higher HSP73 mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Participating in the regulation of folding, assembly and degradation of proteins and protecting cellular proteins from the damage caused by cellular stress like hypoxia or changes in cellular pH, elevated HSP73 expression possibly confers proliferative advantage on melanoma cells.

The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1-10, 10-100, and 100-1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an intenational PCR standard.

Background: Reliable chemosensitivity testing of head and neck squamous cell carcinoma (HNSCC) still faces methodical limitations. Since stromal cell contamination has been found to preclude reliable radiosensitivity testing of HNSCC as well as chemosensitivity testing of lung tumors, the present study investigates the impact of stromal cell contamination on chemosensitivity testing of HNSCC., Patients and Methods: Seventeen biopsies from HNSCC were analyzed. The specimens were investigated using an ex vivo colony formation assay which allows for the quantitative and separate determination of the overall, as well as the epithelial, and stromal response to carboplatin, 5-fluorouracil and docetaxel., Results: The overall chemoresponse was dominated by stromal cell multidrug resistance. However, by selective evaluation of the epithelial chemoresponse, individual chemosensitivity patterns could be identified., Conclusion: Multidrug-resistant stromal cells preclude the reliable assessment of the chemoresponse of HNSCC specimens. Carefiul correction for stromal cell effects is a prerequisite for the generation of therapeutically useful information.

Tumors show an increased glucose uptake that is mediated by glucose transport proteins. We have analyzed the expression of the sodium-dependent glucose co-transporters SGLT-1 and-2 in short-term cultures of squamous cell carcinomas of the head and neck (HNSCC) by RT-PCR. Distribution of the SGLT-1 protein in HNSCC tissues was investigated by immunohistochemistry. While we observed in 17/36 HNSCC short-term cultures the SGLT-1 mRNA, we found no SGLT-2 expression. SGLT-1 mRNA expression occurred preferentially in cultures originating from moderately and well differentiated HNSCC. In tumor tissues a heterogeneous SGLT-1 staining restricted to differentiated tumor cells was seen in 27/30 HNSCC analyzed. In normal mucosa SGLT-1 staining was also confined to differentiated compartments and lacked in dysplastic areas. Our data indicate a differentiation-dependent expression of SGLT-1 in HNSCC. This is important knowledge for the planning of glucose-targeting therapies and suggest SGLT-1 as a differentiation marker in head and neck tissues.

To better understand the correlation of mucosal and systemic immune responses with lentiviral containment, we contrasted the early mucosal and systemic immune responses induced by vaginal versus intravenous exposure of cats to feline immunodeficiency virus (FIV) isolates of differing pathogenicity and clade (i.e., FIV-B-2542 and FIV-A-PPR). We found that despite divergence in viral genotype, the mucosal and systemic immune responses induced differed more with route of exposure than virus isolate. In intravenously exposed cats, Gag-specific antibody (both IgG and IgA isotype) predominated in the serum, saliva, and vaginal wash fluid irrespective of infecting virus isolate. While Env-specific responses were more variable, they were more often detected in vaginally infected cats. Both IgG and IgA directed against Gag and Env were consistently present in vaginal wash fluids independent of route of infection or virus isolate. FIV Gag- and Env-specific cytotoxic lymphocytes (CTLs) were detected in blood and tissue lymphocytes of cats infected with either virus strain but were greatest in intravenously infected animals. Likewise, FIV-specific CTLs were detected in CD8(+) vaginal lymphocytes of animals infected by either route but were also more frequent in intravenously inoculated animals. In summary, we found qualitative differences in the immune responses following vaginal infection but no evidence (1) that mucosal immune responses were enhanced in vaginally exposed cats, (2) that local mucosal infection led to measurably greater immune responses in either compartment; or (3) that more prominent immune responses correlated with lower viral burden.

Approximately 350 Amazon parrots were destined for relocation in Peten province, northeastern Guatemala. In random sampling of the parrots, 95 blood and 75 fecal samples were examined individually for parasites. Coccidia were present in 6.0% (3/50) of Amazona autumnalis autumnalis, and they were the only parasites detected. There were no blood parasites observed in 64 A. a. autumnalis, four Amazona pionus senilis, 16 Amazona ferinosa guatemala, 10 Amazona albifronsus albifronsus, and one Amazona xantholora. No fecal parasites were observed in four A. p. senilis, 12 A. f. guatemala, eight A. a. albifronsus, and one A. xantholora.

Objective: To determine the effect of a commercial bioflavonoid antioxidant on acetaminophen-induced oxidative injury to feline erythrocytes., Design: Randomized controlled study., Animals: 45 healthy age-matched cats., Procedure: Cats were assigned to 3 experimental groups. Groups 1 and 3 received a bioflavonoid antioxidant (10 mg/d) orally for 2 weeks. Groups 2 and 3 received an oxidative challenge with acetaminophen (90 mg/kg [41 mg/lb] of body weight, PO) on day 7. Packed cell volume, percentage of erythrocytes with Heinz bodies, blood methemoglobin concentration, and blood reduced and oxidized glutathione concentrations were determined at various times during the 2-week study period., Results: Adverse effects were not associated with bioflavonoid antioxidant administration alone. Acetaminophen administration resulted in a significant increase in methemoglobin concentration in groups 2 and 3; differences were not detected between these groups. Heinz body concentrations in groups 2 and 3 increased after acetaminophen administration; however, the increase in cats that received the antioxidant was significantly less than in group-2 cats. Total blood glutathione concentrations did not change significantly in groups 2 and 3 after acetaminophen administration; however, ratio of reduced to oxidized glutathione concentration increased significantly after administration in group-2 cats, compared with group-3 cats., Conclusions and Clinical Relevance: Oral administration of bioflavonoid antioxidants to cats at risk for oxidative stress may have a beneficial effect on their ability to resist oxidative injury to erythrocytes.

Regression of feline immunodeficiency virus (FIV) infection was observed in seven of nine vertically infected kittens born to two chronically infected mother cats. Both provirus and nonmaternal FIV antibody were detected in all kittens by 4 weeks of age but only three of the seven kittens were positive by blood mononuclear cell coculture. Between 10 and 14 months of age blood mononuclear cells from each of the seven cats were negative at least once by polymerase chain reaction (PCR), but evidence of virus infection was detected by coculture and/or PCR in biopsied lymph node or bone marrow from five of the seven cats. Despite this evidence of persistent tissue provirus, antibody production did not persist in any of the cats beyond 1 year of age. All seven cats remained asymptomatic although CD4 and CD8 T cell counts were in the low normal range throughout the study. By contrast, two additional perinatally infected littermates that were persistently virus isolation positive developed rapid CD4 depletion and progressed to terminal immunodeficiency by 9 weeks of age. Thus FIV infection can be downregulated and/or sequestered to extremely low levels barely detectable with the assays available, although absolute clearance of virus may not occur. These observations are relevant to human immunodeficiency virus (HIV) infection in paralleling both the apparent "regression" of HIV infection reported in some perinatally infected infants and the low-level, apparently stable, infection established by attenuated simian immunodeficiency viruses.

Mucosal infection by feline immunodeficiency virus (FIV) was assessed via a single exposure of the vaginal or rectal mucosa to either infectious peripheral blood mononuclear cells (PBMCs), infectious plasma, or cell-free cultured virus. All cats inoculated with cell-free cultured virus (100 or 400 TCID) and 9 of 10 cats inoculated with infected PBMCs (2 x 10(7) or 2 x 10(5)) became persistently viremic within 3 weeks. Neither cat inoculated with 2 x 10(3) PBMCs became viremic. Rectal and vaginal exposure were equally effective routes to induce viremia. CD4+ T cells and mitogen-stimulated PBMC proliferation declined in all infected cats. However, a transient PBMC proliferative response to FIV p24gag occurred in most virus-exposed cats, especially those that did not develop detectable infection. FIV was not transmitted by mucosal exposure to infectious virus in plasma (100 TCID), a dose > 10-fold that needed for infection by parental injection. In vitro studies suggested that a plasma heat-stable virus-neutralizing factor may be associated with failure of plasma virus to establish infection via the mucosal route. Mucosal FIV infection provides a new model with which to study early stages of infection and intervention in transmucosal lentivirus infections.

Malignant histiocytosis (MH) was diagnosed in a 13-year-old neutered male Domestic Shorthair cat on the basis of light microscopic and immunohistochemical findings. Thoracic fluid analysis showed a modified transudate which contained a very few atypical discrete cells. Cytologic and histologic evaluation of mediastinal and splenic masses revealed a pleomorphic population of large, discrete, round cells 10 to 30 micrometers in diameter with marked cellular atypia. Nuclei were oval to reniform, often with prominent, bizarre nucleoli. Multinucleated cells and mitotic figures were commonly seen. Erythro- and leucocytophagia were noted. Immunohistochemistry indicated a scattered positive staining pattern with the histiocytic antigenic marker Mac387 and a minor population of cells showing positive reactivity for lysozyme. This report describes the characterization of MH in a cat and emphasizes that MH should be considered as a differential diagnosis in proliferative disorders of discrete-cells in this species.

Invasive cytology of the thoracic and abdominal cavities can provide diagnostic information in a timely manner for the practitioner. The information depends on obtaining a quality sample followed by thorough cytologic evaluation. Diagnostic imaging can enhance the sampling process and minimize the risk. As an adjunct to the historic and clinical information, cytology is valuable in establishing a diagnosis or list of differentials and directing future diagnostics or therapy. The application of cytology of internal organs opens a new window for the differential diagnosis of disease.

We studied vertical transmission of feline immunodeficiency virus (FIV) to determine whether it might provide a model with which to study intervention strategies for mother-to-offspring transmission of human immunodeficiency virus (HIV). We found that pregnant cats acutely infected with FIV (FIV-CSU-2771) transmitted the virus to their offspring via both prenatal and postnatal routes. In utero transmission led to several pathogenic consequences including arrested fetal development, abortion, stillbirth, subnormal birth weights, and birth of viable, virus-infected, and asymptomatic but T cell-deficient kittens. Postnatal milk-borne FIV transmission was demonstrated by the presence of cell-free and cell-associated virus in colostrum and milk and through a foster-nursing experiment. The potential for intrapartum FIV transmission was documented by frequent virus isolation from vaginal wash cells in both the pre- and postpartum periods. FIV transmission was efficient during acute maternal infection, leading to an overall infection rate of 70%. We conclude that FIV vertical transmission may be a useful model with which to evaluate intervention strategies for HIV transmission from mother to child.

A monoclonal gammopathy composed of immunoglobulin G, with concurrent light-chain proteinuria and generalized lymph node plasmacytosis, was associated with chronic pyoderma in a dog. A uniform population of plasma cells was observed cytologically and histologically in multiple lymph node specimens. A diagnosis of monoclonal gammopathy of unknown significance was eventually made by exclusion of other known causes of monoclonal gammopathy, resolution after antibiotic therapy, and no evidence of lymphoproliferative disease after 11 months of follow-up and subsequent necropsy. This report expands the diagnostic considerations for monoclonal gammopathies in the dog.

The effects of 10 mM Ca2+ and Ca2+ channel blockers verapamil, diltiazem and flunarizine on the ouabain-sensitive electrogenic Na+, K+ pump activity of mouse diaphragm muscle fibres enriched with Na+ were compared with the changes in cytosolic [Ca2+]. The electrogenic Na+ pump activity produced by adding K+ to muscles previously bathed for 4 h in a K(+)-free, 2-mM [Ca2+] solution increased the resting membrane potential by about 18 mV. This hyperpolarization was completely inhibited after 10 min incubation in 10 mM Ca2+. Verapamil 10(-5) M, 10(-5) M diltiazem and 10(-7) M flunarizine effectively prevented the effect of elevated [Ca2+]. At these concentrations, these drugs did not affect the K(+)-induced hyperpolarization. In mouse diaphragm, the basal cytosolic [Ca2+] measured by the fluorescent indicator 1-[2-(5-carboxyoxazol-2-yl)-6- aminobenzofuran-5-oxy]2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N '- tetraacetic acid acetoxymethyl ester (fura-2/AM) was 261 +/- 6 nM. After 4 h in a Liley K(+)-free, 2 mM [Ca2+] solution, the cytosolic [Ca2+] increased to 314 +/- 28 nM. Increase in [Ca2+] from 2 to 10 mM caused a twofold increase of cytosolic [Ca2+] to 637 +/- 26 nM. This rise was, like the Ca(2+)-induced inhibition of electrogenic pump, prevented by 10(-5) M verapamil, 10(-5) M diltiazem and 10(-7) M flunarizine. The results suggest that substances which block Ca2+ entry into the cell prevent the Ca(2+)-induced inhibition of the Na+ pump.

Placentas of women suffering from pregnancy-induced hypertension (PIH) were found to contain a greater amount of Na,K-ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K-ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K-ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain-binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis-like factor is present in the body fluids to regulate Na,K-ATPase activity, the present results render its role quite plausible.