Mean Number Of Positive Cells Research Articles

Deletion of 9p21 in urine specimens collected from patients with a history of hematuria and bladder cancer

11038 Background: Fluorescence in situ hybridization (FISH) of voided urine sediment is a sensitive and specific test for the detection of urothelial carcinoma. Common bladder cancer FISH testing consists of 4 DNA probes to chromosomes 3, 7, and 17 and band 9p21. The pattern of enumeration anomalies for these 4 DNA probes are not well documented. We sought to determine the incidence of chromosomal anomalies detected by these 4 probes in urine specimens. Methods: Urine samples from a total of 13,284 patients with a history of hematuria and/or bladder cancer were included for study. FISH was performed in each case using fluorescence-labeled probes to the pericentromeric regions of chromosomes 3, 7, and 17 and band 9p21; cytology was also performed in all cases. Follow-up bladder biopsy information was collected from 280 cases. Results: In total, 91.6% (12163/13284) of urine samples were sufficient for FISH analysis. The overall FISH positive rate was 5.7% (691/12163), and the most frequent patterns of chromosomal anomalies were gains of chromosomes 3 and 7 (56.5%), 7 and 17 (51.6%), and 3 and 17 (36.9%). Deletion of 9p21 was observed in only 12% of cases, and was never observed as the only chromosomal anomaly. The mean number of positive cells in FISH-positive cases was 8.8 (range 4–22). Among 280 cases with matching bladder biopsies, 178 (63.6%) had urothelial carcinoma. FISH was more sensitive than cytology for the detection of urothelial carcinoma (77.4% vs. 53.5%, respectively) (p= 0.03), but there was no significant difference in specificity (68% vs. 71.6%, respectively) (p> 0.05). Conclusions: Gains of chromosomes 3 and 7 by FISH were the most common enumeration anomalies in urine specimens, while deletion of 9p21 only was rare and never seen isolation. FISH was significantly more sensitive than cytology for the detection of urothelial carcinoma with equivalent specificity. No significant financial relationships to disclose.

The Clinical Features and Pathophysiology of Acute Radiation Dermatitis in Patients Receiving Tomotherapy

Radiation therapy (RT) including tomotherapy has been widely used to treat primary tumors, as well as to alleviate the symptoms of metastatic cancers. The primary purpose of this study was to examine the characteristics of the clinical features and pathophysiological mechanisms associated with acute radiation dermatitis in cancer patients that received tomotherapy, and compare the results to patients treated by conventional radiation therapy. The study population consisted of 11 patients that were referred to the dermatology department because of radiation dermatitis after receiving tomotherapy; all patients were evaluated for clinical severity. The patients were assessed and identified using the National Cancer Institute Common Toxicity Criteria version (CTC) 3.0. We performed biopsies of the skin lesions that were examined for apoptosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) assay and stained immunohistochemically with monoclonal antibodies to CD8, CD4 and TGF-beta. As a positive control, patients with radiation dermatitis treated with conventional radiation therapy were also studied. THE RESULTS OF THE CLINICAL FEATURES OF THE SKIN OF TOMOTHERAPY PATIENTS WERE THE FOLLOWING: grade 1 (36%), grade 2 (55%) and other changes (9%). Among the population that had skin lesions due to acute radiation dermatitis, the mean number of positive cells per high power field (HPF) was the following: there were 30.50+/-7.50 TUNEL-positive cells, 34.60+/-12.50 CD8+ T cells, 5.19+/-3.17 CD4+ T cells and 9.95+/-1.33 TGF-beta positive cells measured per HPF. The mean number of positive cells per HPF for the patients that received conventional radiation therapy was: TUNLEL-positive cells in 7.5+/-1.64, CD8-, CD4- and TGF-beta-positive cells in 12.50+/-3.73, 3.16+/-1.47, 6.50+/-1.97. We found that the number of TUNEL-positive cells and CD8+ T cells were higher in the lesions of patients receiving tomotherapy compared to the lesions of the patients receiving conventional radiation therapy. These findings suggest that tomotherapy without dose modification may cause significantly more severe forms of radiation dermatitis by apoptosis and cytotoxic immune responses than conventional radiation therapy.

Mast Cells Regulate the Magnitude and the Cytokine Microenvironment of the Contact Hypersensitivity Response

The role that mast cells play during contact hypersensitivity (CS) response is unclear because some studies have shown that mast cell-deficient mice have relatively intact CS responses whereas others have shown opposing results. Mast cells secrete a wide range of immunomodulatory mediators and can potentially influence the type of immune response generated in the skin during CS. Therefore, we examined the type of microenvironment generated during CS in both W/Wv mast cell-deficient and wild-type mice in response to different immunizing doses of hapten (oxazolone). The CS response elicited after low-dose oxazolone was significantly diminished in W/Wv mice compared with wild-type mice. Unexpectedly, the CS response elicited in W/Wv mice immunized with high-dose oxazolone was more severe compared with wild-type mice. In addition, after immunization with high-dose oxazolone, the granulocyte infiltrate in W/Wv mice was increased by twofold compared with wild-type mice. A shift in the cytokine milieu toward the expression of type-1 cytokines as well as a significant increase in the local adhesion of neutrophils and CD4 T cells in the microvasculature of the skin was observed after hapten challenge in W/Wv mice immunized with high-dose oxazolone compared with wild-type mice. These results suggest that mast cells can act as regulators and inducers of the inflammatory response depending on immunizing stimulus strength.

Cytokeratin 8 and 18 expression in human corneal endothelium

Abstract Purpose: To determine if cytokeratins (CK) 8 and 18 are expressed in healthy corneal endothelium. Methods: Five healthy human corneal discs not suitable for transplantation were used. The mean age of the corneal donors was 70 years (range: 51‐83 years). The corneal buttons were dissected into two parts. One half was used for impression cytology of the endothelium followed by semi‐quantitative RT PCR and immunohistochemical detection of CKs. The second half was frozen and cryosections were prepared. Impression specimens as well as cryosections were fixed and indirect immunofluorescent staining was performed using antibodies against CK8, CK18 (Chemicon) and CK8/18 (Dako). The percentage of positive cells and the intensity of the staining were assessed using fluorescent microscopy. A minimum of two hundred cells were counted per specimen. Results: All impression specimens displayed cells with the hexagonal morphology typical of the corneal endothelium. The mean number of positive cells was 57% for CK8, 8% for CK18 and 42% for CK8/18. The mean number of positive cells in cryosections was 47, 4, and 38% for CK8, CK18 and CK8/18, respectively. Furthermore, expression of the CK8 and CK18 genes was present in all corneas. Conclusions: We detected both CK8 and CK18 in the endothelium of all specimens at both protein and mRNA levels. These results unambiguously demonstrate that cells of the corneal endothelium express the CK pair 8/18 and share some features with simple epithelia.

Cyclosporin A Has a Protective Effect with Induced Upregulation of Hsp70 and nNOS on Severe Spinal Cord Ischemic Injury in Rabbits

The purpose of this study is to ascertain the neuroprotective effect of cyclosporin A on the 25-min surgical ischemia model in the spinal cords of rabbits with neuropathological correlation and histoimmunochemical analyses measuring HSP70 and neuronal NOS (nNOS). New Zealand white rabbits were randomly divided into four groups (each n = 8): the C2, C7, Cs2, and Cs7 groups. The C2 and C7 groups underwent a 25-min surgical aortic cross-clamp without intervention and were sacrificed respectively on day 2 and on day 7 postoperatively. The Cs2 and Cs7 groups received cyclosporin A (25 mg/kg) intravenously 15 min after the 25-min cross-clamp and were sacrificed respectively on day 2 and day 7 postoperatively. Neurologic functions were evaluated on postoperative days 2 and 7 using the Tarlov scoring system. Then the rabbits were sacrificed for histopathologic observation. HSP 70 and nNOS stains with TUNEL assay were done for the C2 and Cs2 groups. All rabbits survived the experimental procedure. Tarlov's score for the Cs7 group (2.75 ± 0.89) was significantly higher than that of the C7 group (1.25 ± 1.39) (p <. 05). Tarlov's score of the Cs7 group was also statistically higher on day 7 than on day 2 (p <. 05). Strong correlation between the neurological and histological scorings was found. The TUNEL assay showed that the mean number of positive cells in the C2 group was 17.5 ± 22.6 and in the Cs2 group was 12.5 ± 11.1, but there was no statistically significant difference between the groups. There was more expression of HSP70 and nNOS in the cyclosporin groups than in the ischemia groups. In conclusion, this study demonstrates that cyclosporin A reduces neurological injury in the rabbit model of 25-min spinal cord ischemia. The neuroprotective effect of cyclosporin A against ischemia seems to be related to overexpressions of nNOS and HSP70.

Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of lesions

Stromal myofibroblasts (MF) have the potential to facilitate progression of neoplastic epithelial lesions that could contribute to their biological behavior. To assess immunohistochemically the frequency of stromal MF in different odontogenic cysts and tumors and correlate it to their aggressive biological behavior. The study included cases of dentigerous cyst (DC, n = 7), odontogenic keratocyst-parakeratinized type (OKC-P, n = 8), orthokeratinized type (OKC-O, n = 9), ameloblastic fibroma/fibro-odontoma (AMF/O, n = 11), unicystic ameloblastoma (UAM, n = 6), and solid ameloblastoma (SAM, n = 7). Cases of oral squamous cell carcinoma (SCC, n = 5) served as control. Myofibroblast frequency was assessed as the number of alpha smooth muscle actin (alphaSMA)-positive stromal cells in 10 high-power fields, presented as the mean number of positive cells per field. Counts showed that mean number of positive cells in OKC-P (25.7+/-11.4) was significantly higher than in DC (8.7+/-11.6) (p = 0.024) and in SAM (29+/-7) it was significantly higher than in UAM (14.9+/-4.9) and AMF/O (5.6+/-7.5) (p < 0.001). Counts in OKC-P and SAM were not significantly different from SCC (21.3+/-5.3) (p > 0.05). The high frequency of stromal MF in known aggressive odontogenic lesions, such as OKC-P and SAM, implies that MF can contribute to the biological behavior of these odontogenic lesions. Various pharmacological agents that control stromal MF can be used as an aid to reduce extensive and mutilating surgery in cases of remarkably aggressive odontogenic lesions.

CpG immunostimulatory sequences enhance contact hypersensitivity responses in mice.

Bacterial DNA and synthetic cytidine-phosphate-guanosine-oligodeoxynucleotides (CpG ODN) potently activate dendritic cells (DC) and therefore have been proposed as adjuvants for vaccination strategies. Although CpG ODN are considered as safe adjuvants this study shows that CpG ODN are responsible for enhanced antigen-specific skin inflammatory reactions. We used the murine model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB) in which hapten-specific CD8+T cytotoxic 1 cells are effector cells. Subcutaneous injection of CpG ODN, 1 d before sensitization enhanced the CHS response to DNFB and resulted in increased recruitment of CD8+ T cells at the challenge sites, whereas control ODN injection did not have any effect. This effect was local and not systemic as it was only observed when DNFB was applied at the same site as the CpG motifs. CpG ODN-induced enhancement of CHS was due to increased antigen-presenting cell functions of DC since: (i) CpG ODN-injected skin revealed upregulated expression of major histocompatibility complex class II, CD80, and CD86 molecules and (ii) CpG ODN treatment of DNFB-derivatized DC enhanced the intensity of CHS responses after in vivo transfer. Taken together, the results show that CpG ODN may be responsible for immune side-effects such as worsening of T cell-mediated skin diseases.

CXCR-4 expression on bone marrow CD34+ cells prior to mobilization can predict mobilization adequacy in patients with hematologic malignancies.

To investigate the mechanisms of mobilization and of the factors implicated in the homing of progenitors and possibly understand the reasons for unpredicted mobilization failure, we analyzed CXCR-4 (CD184) expression on bone marrow (BM) CD34+ cells prior to peripheral blood stem cell (PBSC) mobilization in 24 patients affected by hematologic malignancies (non-Hodgkin lymphoma, multiple myeloma, and acute myeloid leukemia). We wanted to determine whether the level of CXCR-4 expressed by hematopoietic stem cells could influence mobilization process and therefore could be considered a predictive factor for mobilization adequacy. These data were also compared with stromal cell function as assessed by colony forming unit-fibroblast (CFU-F) and CFU endothelial cells (CFU-En) assays and stromal layer confluence capacity exhibited by patients' BM cells. In this study, we also compared CXCR-4 expression on CD34+ cells from different sources and at different migration stages specifically bone marrow (BM), steady state peripheral blood (SSPB), fetal cord blood (FCB), cord blood (CB), and mobilized PBSC. Seven (29%) of the 24 patients undergoing mobilization failed to achieve an adequate number of CD34+ stem cells (5 x 10(6)/kg CD34+ cells) and showed a very high expression frequency of CXCR-4 on BM CD34(+) stem cells (mean number of positive cells, 97%) investigated before the mobilization regimen. We also found that high expression intensity per cell for CXCR-4 was associated with lower amounts of mobilized CD34+ cells whereas those patients (17 out of 24 patients, 71%) with lower expression intensity per cell of CD184 on BM CD34+ cells prior to mobilization harvested at least 5 x 10(6)/kg CD34+ cells. Setting a cut off of 5 x 10(6)/kg CD34+ cells harvested, patients mobilizing less had a mean value of 97% CD34+ cells expressing CXCR-4 with a relative mean channel fluorescence of 458 whereas patients mobilizing more than 5 x 10(6)/kg CD34+ progenitors showed a mean value of 59.8% CD34+/CXCR4+ cells with a relative mean channel fluorescence value of 305. Interestingly, in the poor mobilizers group, the marrow stromal microenvironment was found to be more severely damaged in comparison with that of good mobilizers. The comparative analysis of CXCR-4 expression showed no difference in percentage values between steady-state PB (87.4%) and BM (85.1%) stem cells whereas mobilized CD34+ stem cells have a lower expression frequency of CXCR-4 (71.6%) compared to that of progenitors from other sources. Fetal blood CD34+ stem cells had the lowest mean expression frequency of CD184 antigen (36.3%), while CB cells had the highest (94.8%). In conclusion, this study provides evidence that monitoring CXCR-4 CD34 double positive cells before mobilization can be regarded as a predictive factor for mobilization outcome, giving us directional cues for the choice of the best stem cell mobilization regimens.

Prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in colorectal cancer

The purpose of this study was to evaluate the prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in patients with colorectal cancer. A total of 97 adenocarcinomas of the colon and rectum were examined. Immunohistochemical staining was performed by the standard avidin-biotin-peroxidase complex method using antibodies to CD8 and CD68. Peritumoral infiltration by CD8+ T cells or macrophages was evaluated along the invasive margin of the cancer in each specimen. The area with the most abundant infiltration was selected, and the number of immunoreactive positive cells counted at x400 magnification. Patients were divided into two groups based on the degree of infiltration by each cell type: namely those with a high level of infiltration (more than the mean number of positive cells) and those with a low level of infiltration (less than the mean number of positive cells). Patients with a low level of macrophage infiltration had a significantly deeper depth of invasion than patients with a high level of macrophage infiltration (P=0.027). The percentage of patients with a high level of macrophage infiltration was significantly higher in vascular invasion-negative cases (46.7%) than in vascular invasion-positive cases (22.7%; P=0.045), and in lymph node metastasis-negative cases (52.9%) than in lymph node metastasis-positive cases (28.3%; P=0.014). Overall survival was significantly shorter for patients with a low level of CD8+ T cell infiltration than those with a high level of CD8+ T cell infiltration (P=0.01). The survival rate for patients with a high level of both CD8+ T cell and macrophage infiltration was 100%. In conclusion, both CD8+ T cell and macrophage peritumoral infiltration indicates anti-tumoral action in patients with colorectal cancer.

Proliferation markers, proliferating cell nuclear antigen, Ki67, 5-bromo-2'-deoxyuridine, and cyclin D1 in mouse olfactory epithelium.

We immunohistochemically identified proliferating cells in the olfactory epithelium of mice, using an anti-proliferating cell nuclear antigen (PCNA) antibody, an anti-Ki67 antibody, an anti-5-bromo-2'-deoxyuridine (BrdU) antibody, and an anti-cyclin D antibody. Positive cells stained by the 4 antibodies were identified mainly in the basal layer. The mean numbers of positive cells stained by the 4 antibodies in 500 olfactory epithelium cells from each animal were as follows: PCNA-positive cells 42, Ki67-positive cells 23, BrdU-positive cells 13.7, and cyclin D1-positive cells 9.2. PCNA may be detected in both proliferating and resting cells. Ki67 is an intranuclear antigen expressed in proliferating, but not resting, cells. Anti-BrdU antibodies might stain proliferating cells only following the S-phase, but not the G1-phase. Cyclin D1 is a protein that works during the G1-phase of the cell cycle. When we stain proliferating cells using proliferating cell markers, it is important to consider the cell cycle phases during which each marker stains.

Topoisomerase IIα Expression in Breast Cancer: Correlation with Outcome Variables

Topoisomerase IIα (topo IIα) plays a key role in DNA replication and is a target for multiple chemotherapeutic agents. In breast cancer, topo II expression has been linked to cell proliferation and HER2/neu protein overexpression. However, its relationship with outcome variables is not well established.Formalin-fixed, paraffin-embedded primary breast cancers from 184 women (mean age, 60 years) were stained for topo II by automated immunohistochemistry. A topo II expression index (TI) was determined by counting the number of positive cells per high-power field and calculating an overall mean number of positive cells per high-power field. Tumors with a TI of more than 1 were considered positive, and those with a TI of 1 or less were considered negative. A cell proliferation index was determined by automated immunohistochemistry using the MIB-1 antibody in an identical technique. HER-2/neu gene amplification (HER-2 amp) was determined by automated fluorescence in situ hybridization using the Ventana unique sequence probe.Fifty-nine (32%) of the tumors had a TI greater than 1. On univariate analysis, increased topo II expression correlated with decreased patient survival (p =.001), advanced tumor stage (p =.034), lymph node metastasis (p =.018), and HER-2 amp (p =.016). Tumor stage (p <.0001), node-positive status (p <.0001), tumor grade (p =.025), HER-2 amp (p <.0001), and MIB-1 overexpression (p =.002) also correlated with survival on univariate analysis. Topo II expression did not correlate with tumor size, grade, estrogen receptor/progesterone receptor status, or disease recurrence. On multivariate analysis, stage (p <.0001), lymph node metastasis (p <.0001), and tumor grade (p =.002) all independently predicted disease-related death.Increased topo II expression is associated with an aggressive form of breast cancer featuring HER-2 amp and predicts disease-related death, lymph node metastasis, and advanced tumor stage.

CONSTITUTIVE NF-κB LEVELS IN HUMAN ALVEOLAR MACROPHAGES FROM NORMAL VOLUNTEERS

Alveolar macrophages regulate the inflammatory and immune responses within the lung through cytokine production. Nuclear factor kappa B (NF-κB), a transcription factor, controls the synthesis of cytokines such as interleukin 1β, tumour necrosis factor α, and interleukin 8. In quiescent cells, NF-κB is located in the cytosol as a dimer of protein components (p50, p65) bound to an inhibitor (IκB). Upon activation, NF-κB translocates to the nucleus and binds to DNA. To determine the constitutive level of NF-κB activation in non-smoking normal volunteers, immunohistochemical analysis of alveolar macrophages from 29 subjects was performed with antibody directed against the p65 component of NF-κB. These results were confirmed in four subjects by electrophoretic mobility shift assay (EMSA). A human monocytic cell line, THP-1 with and without endotoxin stimulation was used as positive and negative controls, respectively.The mean number of positive cells was 4.1%±0.8. EMSA performed on whole cell extracts from four normal volunteers demonstrated minimal constitutive binding compared to the positive control. Supershift assay revealed the presence of the p65 dimer. By both immunohistochemistry and EMSA, alveolar macrophages from healthy non-smoking individuals demonstrate minimal NF-κB activation. Immunohistochemistry is a sensitive and quantifiable technique requiring only a minimal number of cells, and this technique may be useful in monitoring small changes in NF-κB activation in inflammatory diseases of the lung.

MIB-1 and PC-10 immunostaining for the assessment of proliferative activity in primary acquired melanosis without and with atypia

AIMSTo compare the proliferative activity of intraepithelial melanocytes in primary acquired melanosis (PAM) without atypia and PAM with atypia by immunohistochemical staining for the Ki-67 antigen and the proliferating cell...

Proliferating cell nuclear antigen in gallbladder carcinoma.

Proliferating cell nuclear antigen (PCNA) expression was studied in 103 gallbladder carcinomas and 23 metastatic lesions as well as in 25 control non-neoplastic gallbladder specimens. Positive nuclear staining was observed in 88% of controls, in 92% of carcinomas and in 70% of metastases. The mean number of positive cells was 21.2% in controls, 44.1% in primary carcinomas and 32% in metastatic cancer cells. Differences which were significant were control v. primary tumour, P < 0.000001; control v. metastasis, P < 0.01 and primary tumour v. metastasis, P < 0.006. In 57 (60%) of the primary tumours there was positive staining in over 40% of tumour cells. We were not able to demonstrate any relationship between macroscopic or microscopic features and PCNA expression. However, tumours confined to the mucosa expressed PCNA more frequently than did more advanced tumours.

Activation of cytotoxic cells in hyperplastic lymph nodes from HIV-infected patients

Serine esterase B (SE B) is a protein contained in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells; SE B gene is transcribed upon activation of these cytotoxic cells. In order to show the in vivo interactions between HIV-infected cells and anti-HIV cytotoxic cells we analysed, by in situ hybridization, the expression of the SE B gene in eight hyperplastic lymph nodes from HIV-1-infected patients presenting with persistent generalized lymphadenopathy. We detected numerous cells expressing the SE B gene. The mean number of positive cells was 3.2 times higher in HIV lymph nodes than in six non-HIV hyperplastic lymph nodes studied in parallel (P less than 0.05). In control lymph nodes, the SE B gene was expressed only in interfollicular areas; virtually no cells expressed the SE B gene within follicles. In contrast, in HIV lymph nodes cells expressing the SE B gene were distributed either in interfollicular areas or within follicles. Expression of the SE B gene inside follicles was thus a specific feature of HIV lymph nodes (P less than 0.001) and was associated with the presence of HIV antigens and RNA at the same site. These results suggest that cytotoxic cells are activated in follicles of HIV lymph nodes and may be involved in the lysis of HIV-infected cells. Such a phenomenon may explain the development of follicle lysis, a specific feature of HIV lymph nodes. It may also inhibit the spreading of HIV infection.

Quantitative and morphometric analysis of Langerhans cells in non-exposed skin in renal transplant patients

Renal transplant recipients have a high incidence of cutaneous complications such as neoplasia and viral or fungal infections. Morphologic alterations of epidermal Langerhans cells (LC) have furthermore been described in these patients. Since these changes have been mainly found in sun-exposed skin, a direct effect of immunosuppressive therapy remains a matter of discussion. A quantitative and morphometric study of epidermal LC in non-exposed skin was performed in 28 renal transplant patients (RTP). RTP were divided in two groups according to immunosuppressive treatment: group A; azathioprine + prednisone (14 cases) and group B; cyclosporine + prednisone (14 cases). Twenty sex-age matched non-immunosuppressed patients acted as controls (group C). Epidermal sheets were obtained by incubation in EDTA and stained for ATPase activity and with the monoclonal antibody T6 (CD1) using the avidin-biotin peroxidase method. Langerhans cells were counted using a calibrated graticule (400x) and expressed as the mean number of LC/mm2. The mean area of the LC and the number of primary dendrites (pd) and secondary dendrites (sd) were determined with a morphometer adapted to an Apple II computer. The mean number of positive cells in controls was: ATPase, 677 +/- 157; T6, 695 +/- 164. Patients in group A had the maximum reduction in both ATPase and T6 LC density (ATPase, 339 +/- 142; T6, 402 +/- 194). Patients in group B had an intermediate reduction in the number of LC (ATPase, 494 +/- 121; T6, 529 +/- 112).(ABSTRACT TRUNCATED AT 250 WORDS)