Diversity and anaerobic growth of Pseudomonas spp. isolated from modified atmosphere packaged minced beef.

AIMS: This study aimed to monitor development of spoilage-associated microbiota on high-oxygen modified atmosphere packaged (MAP) minced beef, assess diversity of Pseudomonas sp. therein employing a polyphasic approach and probe their ability to grow anaerobically in the presence of carbon dioxide. METHODS AND RESULTS: Headspace atmosphere and total viable count of MAP minced beef were monitored, and spoilage-associated microbiota was identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Pseudomonas spp. represented a major part of the spoilage-associated microbiota throughout the spoilage process and were characterized with a polyphasic approach including MALDI-TOF, randomly amplified polymorphic DNA biotyping, 16S rDNA and rpoD sequence analysis, and carA multiplex polymerase chain reaction. Pseudomonas isolates displayed high diversity and varying assertiveness under conditions employed in MAP minced beef with P. fragi, P. lundensis and P. weihenstephanensis as dominant species. CONCLUSIONS: The polyphasic approach enabled high-throughput characterization of Pseudomonas sp. Their adapted capability to grow anaerobically and resistance to high levels of CO2 is suggested to be a general feature within the genus, which is hitherto underexplored. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that diverse Pseudomonas generally regarded as strict aerobes and CO2 -sensitive appear well adapted to grow under MAP conditions, leading to high cell counts in minced beef and ultimately contribute to spoilage of the product.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2.

Recently, proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL). A prominent feature of B-CLL cells is the high expression of CD23, which is closely related to cell survival and is regulated by Notch2. Since several components of the Notch signaling cascade are tightly regulated by proteasomal degradation, we studied the effect of PI on Notch2 activity and CD23 expression. Exposure of B-CLL cells to PI led to induction of apoptosis, a time- and dose-dependent downregulation of CD23 expression and a decline in DNA binding of transcriptionally active Notch2. In contrast, the transcription factor NF-AT and its putative target gene CD5, which is highly expressed in B-CLL cells, were unaffected. When the late phase of PI-induced apoptosis was arrested by inhibition of caspase 3, the reduction of Notch2 activity was still observed, indicating that reduction of active Notch2 took place already during an earlier phase of apoptosis. Enforced expression of constitutively active Notch2 decreased PI-mediated apoptosis in a human B-cell line. These data indicate that downregulation of CD23 and loss of Notch2 activity are early steps in PI-induced apoptosis of B-CLL lymphocytes and may be part of the full apoptotic response.

Reconstitution of endogenous interferon a by recombinant interferon in hairy cell leukemia.

Recombinant human IFN alpha (rhIFN-alpha) plays an important role in the treatment of hairy cell leukemia (HCL). However, the mechanisms leading to its beneficial effect are not completely clarified, and there is no information on IFN-alpha gene expression in this disease. Therefore, we investigated the pattern of IFN-alpha gene expression and protein production in HCL and their potential regulation by rhIFN-alpha. Blood samples from 10 patients with HCL and 8 healthy donors (HD) were investigated. Expression of IFN-alpha mRNA was assessed by reverse transcription-PCR analysis in peripheral blood mononuclear cells (PBMCs) under basal conditions and on induction with rhIFN-alpha and polyionosinic-polycytidylic acid [poly(I.C)]. IFN-alpha concentrations in plasma and culture supernatants were measured by immunoassays, and intracellular IFN-alpha was evaluated by fluorescence-activated cell sorting analysis. Results showed that, in contrast to blood samples from HDs, freshly isolated PBMCs from un treated HCL patients did not express IFN-alpha mRNA, whereas IFN-alpha transcripts were found in patients who were under rhIFN-alpha therapy Plasma of untreated patients contained no, or extremely low levels of IFN-alpha as compared with plasma of treated patients and HDs. Ex vivo treatment of PBMCs with rhIFN-alpha or poly(I.C) resulted in a remarkable up-regulation of IFN-alpha at the mRNA and protein level. In HCL, however the amounts of IFN-alpha protein remained less than in HD. Inhibition of IFN-alpha transcription was found after exposure of PBMCs to serum fron untreated patients. Finally, a reduced capacity to produce IFN-alpha was found within B- cell, T-cell, and monocyte compartments in HCL patients which could be enhanced by rhIFN-alpha. The results demonstrate the ability, of rhIFN-alpha to up-regulate the expression of IFN-alpha gene and protein production and suggest that priming the production of endogenous IFN-alpha is a critical step in the mechanism of action of rhIFN-alpha in HCL.

Current value of double-contrast pharyngography and of computed tomography for the detection and for staging of hypopharyngeal, oropharyngeal and supraglottic tumors.

In light of recent endoscopic techniques the current value of double-contrast pharyngography (DCP) and of CT for detection and staging of hypo-, oropharyngeal, and supraglottic tumors is evaluated. The DCP of 151 patients and CT obtained from 99 of these patients were retrospectively analyzed in a double-blinded manner. We used a standard protocol which comprised all relevant anatomical subregions. Results were compared with direct microlaryngoscopy (DL), indirect laryngoscopy (IL), and post-operative histopathological findings. Sensitivity and specificity of DCP was 75.0 % and 86.7 %, respectively. The DCP and IL techniques together yielded a higher sensitivity (96.7 %) than each method separately. Sensitivity and specificity of CT was 87.5 and 100 %, respectively. In 74.7 % CT provided correct staging. Subregional analysis revealed that the results of DCP and CT depend highly on the localization of the tumor. Our results indicate that DCP represents an important screening method for diagnosing hypo-, oropharyngeal, and supraglottic tumors to complete IL and DL. We show that CT is a reliable method for preoperative staging, although small superficial tumors may occasionally be missed by this method.

Basic fibroblast growth factor is expressed by CD19/CD11c-positive cells in hairy cell leukemia.

Several features are characteristic for hairy cell leukemia (HCL). Among those are pancytopenia, bone marrow fibrosis, and the appearance of a defined tumor cell phenotype in peripheral blood (PB), bone marrow (BM), and spleen. Hairy cells (HC) coexpress antigens specific for B lymphocytes and monocytes/macrophages and thus the malignant cell does not seem to be restricted to a defined lineage. When serum or bone marrow aspirate was screened by enzyme-linked immunosorbent assay (ELISA) for basic fibroblast growth factor (bFGF), specimen derived from HCL (serum: mean value, 29 pg/mL; BM aspirate: mean value, 641 pg/mL) contained significantly higher levels than those from healthy subjects. To study whether peripheral blood mononuclear cells (PBMC) derived from patients suffering from HCL and healthy donors (HD) were capable of producing bFGF, culture supernatant (conditioned medium, [CM]) was tested for the presence of this cytokine. While bFGF was not detectable in cell cultures from HD, HCL-derived CM contained relatively high levels of bFGF. CM was successfully used for stimulation of mesenchymal cell proliferation, which could be inhibited by a neutralizing anti-bFGF antibody. Cellular activation by pokeweed mitogen (PWM) or the combination of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) plus calcium ionophore (Ca-Ip) led to an enhanced mRNA expression. Results of Western blot experiments showed that HC synthesize at least three isoforms (approximately 18, 23, and 25 kD), but only the 23-kD isoform is exported. To assess the nature of the producer cell, double immunofluorescence analysis using a bFGF-specific and an anti-CD11c monoclonal antibody (MoAb) was undertaken. The majority of cells scoring positive for CD11c were also reactive with the anti-bFGF MoAb. Furthermore, enrichment of CD19/CD11c-positive cells correlated with enhanced bFGF levels, thereby supporting the argument for HC being the producer cells of bFGF. A biological function of bFGF in HCL might be mediation of chemoresistance, as 2-chlorodeoxyadenosine (2-CdA)-induced inhibition of cell proliferation can be reversed by bFGF. Endogenous bFGF production by HC is not affected by this purine analogue and 2-CdA-induced apoptosis is diminished in bFGF-producing HC as compared with normal PBMC. Therefore, bFGF expression by HC might be important for resistance to chemotherapy and survival of the malignant cells.

[An immunohistochemical study of the steroid hormone receptors in endometrial hyperplasia]. / Studiul imunohistochimic al receptorilor hormonilor steroizi în hiperplaziile endometriale.

AIM: Evaluation of the estrogen and progesterone receptors (ER and PR) in different degrees of endometrial hyperplasias. METHODS: 10 cases of each degree of endometrial hyperplasia (simple, complex and atypical) were analyzed using the avidin-biotin-peroxidase technique and monoclonal antibodies applied to formalin-fixed, paraffin-embedded tissue. RESULTS: We found similar, high level of both ER and PR in simple and complex hyperplasias and a significant decrease of these in atypical hyperplasia. CONCLUSIONS: Endometrial hyperplasias develop in a setting of estrogen excess. This explains the high level of endometrial receptors and the response to progesterone in typical hyperplasia. In atypical hyperplasia, the decrease of steroid receptors results in a low sensibility of this lesion to progestative therapy, but there are cases with high receptor levels which could have a chance for hormonal therapy.

[p53 and steroid receptors support double endometrial carcinogenesis]. / p53 si receptorii steroizi sustin o dubla carcinogeneza endometriala.

AIM: This study investigated the p53 positivity correlated with the receptor status, in different grades and stages of endometrial carcinoma. METHODS: The study included 88 endometrioid-type carcinomas and 5 non-endometrioid-type carcinomas. Paraffin sections were used for the ordinary histological diagnosis and for immunohistochemical diagnosis by avidin-biotin-peroxidase technique. RESULTS: p53 positivity was identified in 10 endometrioid-type carcinomas (11.4%) and 3 non-endometrioid type carcinomas (60%). Most of the p53 positive cases were receptor negative and observed in advanced stages and histological grades. No associated endometrial hyperplasia was p53 positive, while a positive intraepithelial non-endometrioid carcinoma was identified. CONCLUSIONS: p53 positivity is most frequently identified in non-endometrioid type, receptor negative adenocarcinomas, even in a precursor lesion as it is intraepithelial carcinoma, while in endometrioid, receptor positive carcinomas it appear in late stages of development, never being identified in precursor lesions.

Colposcopy, cervicography, speculoscopy and endoscopy. International Academy of Cytology Task Force summary. Diagnostic Cytology Towards the 21st Century: An International Expert Conference and Tutorial.

ISSUES: The colposcope was developed in 1925 and is well established in clinical gynecologic practice for defining and delineating cytologically detected lesions mainly of the cervix but also the vagina and vulva. Additionally, various endoscopic procedures in gastroenterology, pulmonary and urologic lesions enhance the cytologic detection and histologic verification of precancerous and cancerous lesions. The cost-effectiveness of all these devices and their applicability, particularly in countries with a limited health budget, is a major issue. This task force considered aspects of the present state of the art and the challenges in the 21st century. CONSENSUS POSITION: Automated cytology can interface with colposcopic examination in a number of significant ways. Automated cytologic analysis of conventional cervical smears can potentially direct colposcopic examination by predicting the nature of a lesion, assist in determining which patients should receive colposcopy and, in some settings, thereby reduce the number of colposcopies. Potentially, various combinations of automated cytology and colposcopy may be used to generate screening protocols that might result in more effective and inexpensive screening. The role of cervicography, or high-resolution cervical photography, as a screening device remains to be defined. Sensitivity for high grade lesions is generally no greater than that in cytology, and specificity appears lower. The interpretation of cervical photographs in triage of mildly abnormal cytology may prove to be useful in countries with established cytology programs. In areas of the world where cytology screening programs are not in place, the interpretation of cervical photographs may have its most dramatic effect. Cost-effectiveness analyses are needed. There are, at present, insufficient data for the evaluation of speculoscopy, a procedure using chemiluminescent illumination of the cervix for visualization of acetowhite areas. Basic training in colposcopy should be integrated into the residency programs of obstetrics and gynecology. Criteria for the adequate training of colposcopists should be developed. Continuing education programs in colposcopy should be developed when they are not already in existence. The cost-effectiveness of integrating colposcopy as a primary screening technique should be evaluated. Following a high-grade squamous intraepithelial lesion (HSIL) cytology result, colposcopically directed punch biopsy should be taken with or without endocervical curettage. This generally should precede the loop electrosurgical excision procedure (LEEP); however, in certain circumstances direct LEEP may be indicated. LEEP under colposcopic vision is an efficient way to treat an HSIL lesion of the cervix because the histologic extent and margins can be determined, unlike with laser surgery or cryosurgery. It is also more cost-effective than cold knife conization because general anesthesia and an operating room are unnecessary. Following LEEP, the endocervical canal should be examined colposcopically for any evidence of involvement. Lesions in the endocervix can then be removed with a different-shaped loop. Further research into Raman spectroscopy as a diagnostic aid in cervical pathology is needed, as is the use of micrococolpohysteroscopy for in vivo cytologic analyses, especially of the endocervical canal and transformation zone. Hysteroscopy is the most direct method for the diagnosis and treatment of intrauterine diseases. Hysteroscopic endometrial biopsy is more accurate than conventional biopsy methods. Cervical invasion of endometrial cancer can be detected by hysteroscopy. The depth of invasion, however, is more accurately determined by magnetic resonance imaging or computed tomography. ONGOING ISSUES: Many topics for ongoing research and/or implementation are mentioned under "Consensus Position," above. (ABSTRACT TRUNCATED)

Inadequate production of hematopoietic growth factors in hairy cell leukemia: up-regulation of interleukin 6 by recombinant IFN-alpha in vitro.

The course of hairy cell leukemia (HCL) is characterized by progressive pancytopenia. The pathogenesis of this phenomenon is still not fully understood. To study if the decrease in hematopoiesis in HCL is accompanied by abnormal concentrations of growth factors, we investigated the production of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin 3 (IL-3), interleukin 6 (IL-6), and tumor necrosis factor alpha by peripheral blood mononuclear cells (PBMCs) of eight patients with HCL. The results point to a severe deficiency of production of all cytokines tested as compared to healthy donors. However, enrichment of autologous monocytes by counterflow centrifugation resulted in a marked increase of the levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-6, and tumor necrosis factor alpha. The most pronounced effects were seen with IL-6. Reverse transcription-PCR analysis indicated that pokeweed mitogen, IFN-alpha, and poly(I:C) are capable of inducing the expression of IL-6-specific mRNA in HCL cells. These findings are substantiated on the protein level by immunofluorescence analysis. Incubation of PBMCs with IFN-alpha resulted in a significant increase of intracellular IL-6 in HCL but not in healthy donors. This increase was also seen in hairy cells positive for CD19 and CDllc. Furthermore, IFN-alpha induced the secretion of IL-6 from PBMCs of HCL patients but not healthy donors. In conclusion, our studies with PBMCs from patients with HCL revealed an inadequate supply of hematopoietic growth factors that might, in part, be due to the monocytopenia characteristic for this disease. The findings also indicate that IFN-alpha is capable of inducing the production of IL-6 in the patients' PBMCs as well as in their hairy cells. These data from our in vitro studies support the clinical observation that treatment with IFN-alpha leads to reconstitution of hematopoiesis.

The colposcopic screening.

The objective of this paper is a valuation of colposcopy as a screening tool. As database, 392 patients with histologically confirmed intraepithelial neoplasia were used. Colposcopic and cytologic findings were compared with the final histologic diagnosis. The following results were obtained: (1)The colposcopic findings correlated with the histologic diagnosis to a significantly higher degree than the cytologic findings. Depending upon the rate of dysplasia, the colposcopic findings predicted the diagnosis in 84-97%. (2) The false-negative rate of cytology in condylomatous lesions and mild dysplasia was high (39 and 26%, respectively), in particular in comparison with the false-negative rate of colposcopy of 5% for both lesions. Thus, a negative smear does not exclude consistently a dysplasia of the cervix. (3) The false-negative rate of cytology for the high grade lesions (CIN II and CIN III) was 13 and 1% respectively and, thus, lower than in the low grade lesions. There were, however, considerable discrepancies in comparison with the histologic rating of the lesion. In CIN III cytology correlated with histology in only 61%, colposcopy, however, 85% (P < 0.001). Our results demonstrate that colposcopy is an excellent tool for detecting HPV caused lesions especially subclinical lesions and CIN I. Colposcopy is also a corrective for the false-negative cyto-smear rate (about 20-40%). Thus, colposcopy may be used as an effective quality assurance method and an excellent screening method in that colposcopy is superior in grading dysplastic lesions of the cervix. The application of the European terminology was advantageous.

DNA flow cytometry in node-positive breast cancer. Prognostic value and correlation with morphologic and clinical factors.

OBJECTIVE: To help clarify the possible usefulness of nuclear DNA content and S-phase fraction (SPF) as additional prognostic factors in node-positive breast cancer patients because there is increased interest in the development of new factors that might provide more detailed prognostic information. STUDY DESIGN: We performed a DNA and SPF analysis by flow cytometry using a multivariate statistical model on a group of 139 node-positive breast cancer patients with clearly defined inclusion and exclusion criteria. RESULTS: The percentage of aneuploidy increased with the number of involved nodes. Aneuploid tumors were more often found among grade 3 and among receptor-negative tumors. Univariate analysis showed a strong effect on recurrence-free survival (RFS) for the number of involved nodes (P < .001) and for tumor size (P = .013). Grade 3 and receptor-negative tumors showed a nonsignificant trend toward increased risk. The relative risk of aneuploid tumors was 1.19 (95% confidence interval, 0.75-1.87). Multivariate analysis revealed only the number of involved nodes to be an independent prognostic factor (P = .002); ploidy showed no effect (P = .684). SPF did not show any significant effect on RFS, even in a univariate analysis. CONCLUSION: These results suggest that nuclear DNA content and SPF correlate with morphologic factors. Their routine clinical use, however, in node-positive breast cancer patients receiving adjuvant therapy seems to have no clinical relevance and therefore can be omitted.

Soluble CD23 reliably reflects disease activity in B-cell chronic lymphocytic leukemia.

PURPOSE: This study was initiated to evaluate whether soluble CD23 (sCD23) reflects disease activity and tumor load in B-cell chronic lymphocytic leukemia (B-CLL) and to determine its prognostic potential for this disease. PATIENTS AND METHODS: The concentration of sCD23 was measured in the serum of 45 B-CLL patients, 50 patients with other lymphoproliferative disorders, and 41 healthy donors (HD). sCD23 serum levels from B-CLL patients were correlated with parameters of disease activity and total tumor mass (TTM) score. In selected cases, sCD23 was measured repeatedly over a 24-month period. Expression, density, and calculated total amount of membrane CD23 on peripheral-blood B-CLL cells, as well as its correlation to sCD23 levels in serum and supernatants, were determined. RESULTS: sCD23 in B-CLL patients serum was highly elevated as compared with other lymphoproliferative disorders, with the exception of immunocytoma (IC). Both advanced Rai stages and active forms of B-CLL were associated with higher levels of sCD23. There was a highly significant reciprocal relationship between sCD23 and lymphocyte count doubling time (LCDT). Serum sCD23 correlated positively with serum deoxythymidine kinase activity and TTM score, but not with absolute lymphocyte counts. The repetitive measurement of serum sCD23 showed the usefulness of this marker in monitoring disease progression in B-CLL. The total amount of membrane CD23 on in vitro-cultured B-CLL cells correlated significantly with sCD23 levels in the supernatant, whereas correlation between serum sCD23 and membrane CD23 on freshly isolated B-CLL cells was absent. CONCLUSION: Our results indicate that sCD23 is a highly sensitive and specific parameter with prognostic potential for B-CLL, which may be used as a tumor marker and may help to assess disease activity.

Myelosuppression in HCL: role of hairy cells, T cells and haematopoietic growth factors.

To elucidate mechanisms which may be responsible for the haematopoietic insufficiency in hairy cell leukaemia (HCL), we investigated in an autologous in vitro system the influence of haematopoietic growth factors (CSFs) and the effects of hairy cells (HCs) as well as T cells on the formation of haematopoietic colonies (CFU). Colony forming assays were performed using peripheral blood mononuclear cells (PBMC) of 6 HCL patients. To remove HCs, PBMCs were subjected to complement-mediated lysis, T cells were removed by E-rosette formation. Assays were done with and without recombinant human (rh) interleukin-3 (IL-3) and rh granulocyte-macrophage-colony-stimulating factor (GM-CSF). All 6 patients exhibited a severe reduction of their circulating progenitor cell (CPC) compartment. There was no correlation between the degree of colony reduction and the number of HCs. However, a correlation was found between the numbers of CPCs of HCL patients and healthy donors and the monocyte counts in these groups (r = 0.8573, p < 0.001). The removal of autologous HCs, but also of T cells, resulted in a significant increase in colony formation (BFU-E, CFU-GM, CFU-mix). In none of the experiments, however, did colony numbers come close to the normal range. This was only achieved by supplementation of the culture medium with rh IL-3 and rh GM-CSF. The results suggest that the haematopoietic failure observed in HCL patients is probably due to an inadequate supply of CSFs as well as to an inhibitory activity of HCs and T cells which might exert their effects in a synergistic fashion. There is also evidence that the lack of monocytes plays a role in the development of the haematopoietic insufficiency in HCL.

Human papillomavirus, type 16, DNA in multicentric anogenital neoplasia associated with idiopathic panmyelopathy. A case report.

A 27-year-old woman suffering from panmyelopathy for six years presented with a cervical low grade squamous intraepithelial lesion (SIL), vulvar high grade SIL and perianal squamous cell carcinoma with an inguinal metastasis. Southern blot hybridization with 32P-labeled human papillomavirus (HPV) DNA revealed HPV 16 DNA in varying copy numbers in material from the four locations. HPV 16 genomes persisting after surgery on the perianal tumor area were no longer detectable after betatron radiotherapy.