Annular Stenotic Oesophageal Squamous Cell Carcinoma in Cattle Exposed Naturally to Bracken Fern (Pteridium arachnoideum).

Oesophageal squamous cell carcinomas (SCCs) may be observed as exophytic masses or ulcerative or infiltrative endophytic neoplasms. However, in cattle, there is also an uncommon gross presentation as an annular stenotic thickening of the oesophageal wall. Thirteen cases of annular stenotic oesophageal SCC in cattle grazing in bracken fern (Pteridium arachnoideum) areas are reported. The lesions consisted of endophytic masses, focally extensive, firm and circumferential (annular) in the oesophageal wall. Pronounced wrinkling of the mucosa, with retracted uneven areas and subsequent luminal narrowing (stenosis), was observed in all cases. Papillomas and squamous intraepithelial lesions also were observed in these cases. The SCCs were graded as well differentiated (n = 7), moderately differentiated (n = 5) or poorly differentiated (n = 1). The neoplastic keratinocytes were surrounded by moderate to abundant fibrous connective tissue (a desmoplastic reaction), that was better demonstrated by Masson's trichrome stain. Picrosirius red-stained sections showed abundant collagen type I fibres, which contributed to the stenosing characteristics of this tumour. Although it might be easily misdiagnosed as oesophageal scar tissue, the oesophageal SCCs of cattle grazing bracken fern may have a distinctive gross appearance that should be included in the differential diagnosis of oesophageal stenosis.

Virological and clinico-pathological features of orf virus infection in experimentally infected rabbits and mice.

Many aspects of the biology of orf virus (ORFV) infection remain poorly understood and attempts to establish animal models have yielded conflicting and non-reproducible results. We herein describe the characterization of ORFV infection and disease in rabbits and mice. A protocol of intradermal inoculation was employed to inoculate 10(8.5)TCID50/mL of ORFV strain IA-82 in the skin of ears, of the back and labial commissures. All inoculated rabbits presented a clinical course characterized by erythema, macules, papules/vesicles or pustules that eventually dried originating scabs. Local signs started around days 3 and 4 post-inoculation (pi) and lasted 3-10 days. Virus was recovered from lesions between days 2 and 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit milder clinical course occurred in 5/10 inoculated mice; virus was recovered from lesions from three animals. Inoculated lambs - used as controls - developed severe lesions of contagious ecthyma. VN tests performed at day 28pi failed to detect neutralizing antibodies in all inoculated animals. In contrast, convalescent rabbit sera were positive by ELISA at dilutions from 100 to 400. These results show that rabbits are susceptible to ORFV infection and thus may be used to study selected aspects of ORFV biology.

Morphological factors as indicators of malignancy of squamous cell carcinomas in cattle exposed naturally to bracken fern (Pteridium aquilinum).

Squamous cell carcinomas (SCCs) of the upper digestive tract (UDT) of cattle have been associated with chronic bracken fern (Pteridium aquilinum) toxicity and infection with bovine papillomavirus type-4. These tumours share some morphological similarities with human head and neck SCCs. In this study, morphological changes were correlated with the biological behaviour of 40 alimentary SCCs in cattle grazing on pastures with high bracken content. The majority of SCCs were localized to the cranial and caudal regions of the UDT (almost 45% each). More than 60% of the tumours were well differentiated and were found mostly in the cranial region. Metastasis occurred in 58% of the cases, mostly to regional lymph nodes. All poorly differentiated SCCs had evidence of metastasis. Morphological patterns characterized by islands and ribbons of neoplastic keratinocytes were more prominent in well differentiated SCCs. These patterns varied greatly in moderately differentiated SCCs. Poorly differentiated tumours were characterized by the presence of cellular aggregates and individual cells and these tumours had more marked desmoplasia. A significant positive association was established between lymphoplasmacytic inflammatory infiltration and tumour-associated tissue eosinophilia. Evaluation of argyrophylic nucleolar organizer regions (AgNORs) revealed higher proliferation indices in poorly differentiated tumours than in moderately or well differentiated lesions. There was significant correlation between the AgNOR index and histological grading. The morphological factors analyzed were all related to histological grading, which is the major factor predicting the biological behaviour of SCCs in cattle naturally exposed to bracken fern.

Ribosomal protein S6 is a selective mediator of TRAIL-apoptotic signaling.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a potent inducer of apoptosis in tumor cells and holds a promise as a therapeutic agent against cancer. To elucidate the death signaling evoked by TRAIL, we performed a functional genetic screening and rescued TRAIL-resistant Jurkat clones harboring ribosomal protein S6 (rpS6) cDNA in anti-sense frame. Reduction of rpS6 expression in Jurkat and HeLa cells attenuated apoptosis induced by TRAIL, but not those by other cell death signals, including tumor necrosis factor-alpha and cycloheximide, etoposide, doxorubicin, tunicamycin and staurosporine. Death receptor (DR) 4, but not DR5, was downregulated in rpS6 knockdown cells. Conversely, the sensitivity to TRAIL was increased by the ectopic expression of wild-type rpS6 and further by phospho-defective rpS6 mutant (S6-SS235,6AA), but not by phospho-mimic rpS6 mutant (S6-SS235,6DD). Also, unphosphorylatable rpS6 knock-in mouse embryo fibroblasts (rpS6(P-/-) MEFs) were more sensitive to TRAIL than control MEFs. In addition, SKHep-1 tumor cells, which express less phospho-rpS6 and are more sensitive to TRAIL than other tumor cells, became effectively desensitized to TRAIL after rpS6 knockdown. These results suggest that rpS6, especially in its unphosphorylated form, is a selective mediator of TRAIL-induced apoptosis.

Phenotypic differences between electrocardiographic and echocardiographic determination of hypertrophic cardiomyopathy in genetically affected subjects.

OBJECTIVES: In the molecular era, two types of phenotypic differences are recognized between electrocardiography (ECG) and echocardiography in hypertrophic cardiomyopathy (HCM); ECG abnormalities in carriers without left ventricular hypertrophy (LVH), and normal ECG patterns in carriers with LVH. The goal of this study was to evaluate the diagnostic value of ECG for detecting carriers without LVH, and also to assess normal ECG patterns in carriers with LVH from the genetic standpoint of HCM. SETTING: A matched case-control study in a university hospital and general hospitals in Japan. PATIENTS AND DESIGN: ECG and echocardiographic findings were analysed in 173 genotyped subjects (107 genetically affected, 66 unaffected) from families with disease-causing mutations in four genes. RESULTS: ECG abnormalities were found in 18 (54.5%) of 33 nonhypertrophic carriers, but only nine (13.6%) of 66 noncarriers (P < 0.001). For detecting nonhypertrophic carriers, ST-T abnormalities showed the highest accuracy amongst the three major ECG criteria. In contrast, normal ECG patterns were found in eight (10.8%) of 74 carriers with LVH. The sensitivity of ECG for detecting carriers with LVH in families with the cardiac myosin-binding protein C, cardiac troponin T and cardiac troponin I gene mutations was 83%, 88% and 94% respectively. CONCLUSION: These findings suggest that ECG may have favourable diagnostic value even for detecting nonhypertrophic carriers. Furthermore, diagnostic value of ECG may differ according to the genes involved. Our data may contribute to interpretation of phenotypic differences between ECG and echocardiography from the viewpoint of molecular genetics of HCM.

Clinical study of urine NMP 22 (nuclear matrix protein 22) as a tumor marker in urinary epithelial cancer.

The prognosis of urinary epithelial cancer is still poor, and early detection of this cancer is strongly desirable. The sensitivity of conventional urinary cytology is not satisfactory enough. It is hoped that a specific tumor marker will be established. In recent years, it has been reported that urine NMP 22 is very useful and that urine BFP is also relatively useful. We have now determined urine NMP22 and BFP and studied their clinical usefulness as a tumor marker. Using patients diagnosed with histologically confirmed urinary epithelial cancer as the subjects, we retrospectively studied the usefulness of NMP 22, BFP and cytology mainly with regard to the sensitivity (positivity rate), and also in relation to atypia, degree of infiltration and clinical course.

Functional screening of genes suppressing TRAIL-induced apoptosis: distinct inhibitory activities of Bcl-XL and Bcl-2.

TNF-related apoptosis-inducing ligand (TRAIL) is known to selectively induce apoptosis in various tumour cells. However, downstream-signalling of TRAIL-receptor is not well defined. A functional genetic screening was performed to isolate genes interfering with TRAIL-induced apoptosis using cDNA retroviral library. Bcl-X(L) and FLIP were identified after DNA sequencing analysis of cDNA rescued from TRAIL-resistant clones. We found that increased expression of Bcl-X(L), but not Bcl-2, suppressed TRAIL-induced apoptosis in tumour cells. Western blot and immunohistochemical analyses showed that expression of Bcl-X(L), but not Bcl-2, was highly increased in human breast cancer tissues. Exposure of MDA-MB-231 breast tumour cells to TRAIL induced apoptosis accompanied by dissipation of mitochondrial membrane potential and enzymatic activation of caspase-3, -8, and -9. However, SK-BR-3 breast tumour cells exhibiting increased expression level of Bcl-X(L) were resistant to TRAIL, though upon exposure to TRAIL, caspase-8 and Bid were activated. Forced expression of Bcl-X(L), but not Bcl-2, desensitised TRAIL-sensitive MDA-MB-231 cells to TRAIL. Similar inhibitory effects were also observed in other tumour cells such as HeLa and Jurkat cells stably expressing Bcl-X(L), but not Bcl-2. These results are indicative of the crucial and distinct function of Bcl-X(L) and Bcl-2 in the modulation of TRAIL-induced apoptosis.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

NFATz: a novel rel similarity domain containing protein.

Nuclear Factor of Activated T cell (NFAT) is a family of transcription factors that are important for the coordinate expression of various cytokines and immunoregulatory cell surface molecules in T cells and other types of cells in the immune system. In addition, analysis of gene disrupted mice revealed that some members of NFAT family are important for the development of myocardium, myocardial hypertrophy, and mesenchymal stem cells. NFAT family proteins have two conserved domains, the NFAT Homology Domain (NHD) and the Rel Similarity Domain (RSD). The RSD is DNA binding and AP-1 interacting domain which has structural similarity to the Rel Homology Region, the DNA binding domain of Rel family proteins. The NHD is a regulatory domain required for the Ca regulated translocation of NFAT. We report here the isolation and initial characterization of a novel RSD containing protein designated NFATz. NFATz has a RSD but no NHD. NFATz protein is localized in the nucleus without Ca signal. There is no detectable binding to a typical NFAT site even in the presence of AP-1, and it is not capable of activating transcription through the NFAT site. The chromosomal location determined by FISH revealed that NFATz and NFATx genes are in the same region.

Risk factors for venous thromboembolism following prolonged air travel. Coach class thrombosis.

Venous thromboembolism (VTE) in legs and lungs is a potentially life-threatening condition. The incidence of VTE associated with air travel is still unknown, but it may have increased. Most travelers who develop symptoms do so within 24 hours after their flight takes off. Predisposing risk factors may be divided into patient-related and cabin-related factors, both of which are described. It is emphasized that better information and better inflight precautions can minimize these risk factors.

Indications for surgical treatment of iliofemoral vein thrombosis.

The goals of treatment of acute iliofemoral DVT should be prevention of fatal PE, reduction of pain and swelling of the involved leg, trying to stop the development of phlegmasia cerulea dolens and venous gangrene, prevention of disabling PTS by early removal of the blood clot, avoiding proximal venous obstruction, preserving normal, functioning valves in the leg veins, and preventing reflux. The authors recommend an aggressive approach with rapid removal of the occluding thrombus in the leg veins extending up into the iliac veins in active patients with a short history of symptomatic DVT, usually less than 7 days. This approach is not justified in chronically ill, bedridden, high-risk, or aged patients, or those with serious intercurrent disease or limited life expectancy. In these patients, such interventions can only be indicated for limb salvage in phlegmasia cerulea dolens when conservative treatment does not prevent the development of an acute compartment syndrome with venous gangrene. The preferred means of accomplishing early and quick removal of the thrombus is CDITT. Most of the authors' positive experience with thrombolysis is based on the use of urokinase. The Food and Drug Administration (FDA) has put this drug on temporary hold for almost 1 year. The alternative drugs (e.g., tissue plasminogen activator [tPA]) have not been tested for CDITT of DVT, and tPA is not FDA-approved for this indication. When there are contraindications or failure of thrombolysis, TE with a temporary AVF is a valid alternative.

Rab37 is a novel mast cell specific GTPase localized to secretory granules.

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.

ret/PTC expression may be associated with local invasion of thyroid papillary carcinoma.

BACKGROUND AND OBJECTIVES: The exact role of ret/PTC in the development of papillary carcinoma remains unclear. Expression of the ret/PTC oncogene was examined immunohistochemically to address its role in the progression of thyroid carcinomas. METHODS: Paraffin-embedded samples from 34 clinically evident thyroid papillary carcinomas and 19 occult papillary carcinomas were analyzed using an antibody raised against the ret tyrosine kinase domain. RESULTS: Expression of ret/PTC was demonstrated in 6/19 (32%) occult carcinomas. The frequency of expression of ret/PTC in clinically evident carcinomas in 16/34 (47%) was significantly higher than in normal tissues (0%) and follicular adenomas (1/14, 7%, P < 0.01).ret/PTC expression was observed more frequently in the peripheral areas of clinically evident carcinomas (P < 0.01). Although there was no correlation of ret/PTC expression with tumor size, lymph node metastasis, or distant metastasis, the incidence of ret/PTC expression in tumors with extrathyroidal invasion (13/19, 68%) was significantly higher than those without extrathyroidal invasion (3/15, 20%, P < 0.01). Local invasion was found in none of the occult carcinomas. The frequency of expression in occult carcinomas was significantly lower than in clinically evident carcinomas with extrathyroidal invasion (P < 0.05). CONCLUSIONS: The ret/PTC oncogene may be involved in the local invasion of thyroid papillary carcinomas.

Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.

BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.

Two independent calcineurin-binding regions in the N-terminal domain of murine NF-ATx1 recruit calcineurin to murine NF-ATx1.

Intracellular calcium regulates events controlling nuclear translocation of nuclear factor of activated T cells (NF-AT). Calcium-dependent phosphatase calcineurin (CN) plays a central role in this process. Structural and functional analyses of the N-terminal domain of murine NF-ATx1, a member of the NF-AT family, have defined two distinct CN binding regions (CNBRs), CNBR1 and CNBR2, which are located in the region preceding the SP boxes of serine/proline-rich sequences and the region between the SP boxes and Rel similarity domain, respectively. The binding of murine NF-ATx1 (mNF-ATx1) to CN was abolished by deletion of these two regions, yet was unaffected by the individual deletion. In contrast, the nuclear translocation of mNF-ATx1 was much reduced when only CNBR2 was removed. Luciferase assay revealed that both regions are required for mNF-ATx1-dependent activation of the murine IL-2 promoter. Most importantly, recombinant CNBR2 bound CN with a higher affinity, and when expressed in Jurkat cells, it functioned as a dominant negative mutant that prevented the transcription driven by exogenous mNF-ATx1, probably by interfering with the function of CN. We propose that activation of mNF-ATx1 can be modulated through two distinct CN target regions. Our findings provide a new opportunity for pharmacological intervention with Ca2+-dependent signaling events.

Late recurrence of acinic cell carcinoma of the parotid gland.

Acinic cell carcinoma of the salivary glands is a rare cancer representing a low grade malignancy. The recurrence of a tumor is sometimes encountered, usually within 5 years of initial operation. We describe an unusual recurrence after a long interval following primary surgery. In 1987, a 60-year-old woman underwent excision of a mass in the superficial lobe of the right parotid gland under the preoperative diagnosis of a benign tumor. A histologic diagnosis of acinic cell carcinoma was made by examining sections from the resected mass. The patient noted several small nodules in the right parotid region in 1995, but she did not visit our clinic until 1998 when tenderness developed. A locally recurrent tumor and cervical lymph nodes containing metastases were resected and postoperative radiotherapy was given 11 years after the first operation. At least 10 years of follow-up may be necessary for patients with acinic cell carcinoma because of slow-tumor growth.

Signalling into the T-cell nucleus: NFAT regulation.

The nuclear factor of activated T cells (NFAT) plays an important role in T-cell biology. Activation of T cells results in the rapid calcineurin-dependent translocation of NFAT transcription factors from the cytoplasm to the nucleus. This translocation process coupled to the subsequent active maintenance of NFAT in the nucleus compartment is critical for the induction of expression of several genes encoding cytokines and membrane proteins that modulate immune responses. The molecular cloning of the NFAT family of transcription factors has facilitated rapid progress in the understanding of the signalling mechanisms that control the activity of NFAT.

Venous bypass and valve reconstruction: long-term efficacy.

Palma and Esperon described the first femoro-femoral cross-over bypass for iliac vein obstruction in 1958, and Kistner performed the first valve reconstruction for deep vein reflux in 1968. Such surgical development has stimulated better diagnostic methods that now form the foundation for a classification of chronic venous disease, and surgery has been supported by, and sometimes replaced by, the rapid progress in endovascular procedures with angioplasty and stenting. The ability now exists to relieve obstruction and repair reflux in the deep veins, and the results in the successful cases demonstrate the improvement that follows correction of the physiologic abnormalities. The detail in workup required to achieve an accurate diagnosis that is adequate enough to guide surgical treatment in these cases has set a new standard for the diagnosis of chronic venous disease that incorporates the clinical state, etiology, pathophysiology and anatomic distribution of the venous problem, and is incorporated in the CEAP (clinical, etiologic, anatomic, pathophysiologic) classification. The challenge at this time is to produce a reliable set of data that demonstrate the results of treatment in patients with chronic venous disease by conventional methods of bandaging, rest and elevation as well as specific surgical correction of venous obstruction and reflux and to follow these cases over a significant period of time.