Attenuated Toxoplasma gondii ts-4 mutants engineered to express the Leishmania antigen KMP-11 elicit a specific immune response in BALB/c mice.

In order to test recombinant Toxoplasma as adjuvant and live vaccine carrier in the infectious disease model of murine experimental leishmaniasis, we engineered the attenuated, temperature-sensitive Toxoplasma gondii strain ts-4 to express the heterologous Leishmania antigen kinetoplastid membrane protein-11 (KMP-11). Transgenic ts-4 clones were obtained which express KMP-11 as cytoplasmatic protein or target it to the secretory pathway of the tachyzoites. Immunization of BALB/c mice with these stably transformed parasites elicited proliferative responses to both T. gondii antigen and recombinant KMP-11. When challenged with Leishmania major, we observed significant protection in animals that had been vaccinated with the KMP-11-expressing ts-4 mutants. The adjuvant attenuated only the onset of the Leishmania infection, but animals were ultimately not able to control the disease. Thus, our findings demonstrate that recombinant Toxoplasma has the potential to serve as an efficient vaccine carrier for cutaneous leishmaniasis. Furthermore, they establish a protective role for the antigen KMP-11 when given in such a vaccine formulation.

General purpose, field-portable cell-based biosensor platform.

There are several groups of researchers developing cell-based biosensors for chemical and biological warfare agents based on electrophysiologic monitoring of cells. In order to transition such sensors from the laboratory to the field, a general-purpose hardware and software platform is required. This paper describes the design, implementation, and field-testing of such a system, consisting of cell-transport and data acquisition instruments. The cell-transport module is a self-contained, battery-powered instrument that allows various types of cell-based modules to be maintained at a preset temperature and ambient CO(2) level while in transit or in the field. The data acquisition module provides 32 channels of action potential amplification, filtering, and real-time data streaming to a laptop computer. At present, detailed analysis of the data acquired is carried out off-line, but sufficient computing power is available in the data acquisition module to enable the most useful algorithms to eventually be run real-time in the field. Both modules have sufficient internal power to permit realistic field-testing, such as the example presented in this paper.

A genetically engineered cell-based biosensor for functional classification of agents.

Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agent's ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).

Absence of type I estrogen receptors in choroidal melanoma: analysis of Collaborative Ocular Melanoma Study (COMS) eyes.

PURPOSE: To evaluate choroidal melanomas in enucleated eyes for the presence of type I estrogen receptors. METHODS: Fourteen consecutive eyes with large choroidal melanomas (defined as >16-mm basal diameter and >8 mm thickness) from 14 patients (eight women and six men with a mean age of 57 years; range, 25--74 years) enucleated in accordance with the Collaborative Ocular Melanoma Study (COMS) protocol were investigated. Immunohistochemical techniques were employed to label the choroidal melanomas for the presence of type I estrogen receptors. Each specimen was then evaluated in a masked fashion by an experienced ophthalmic pathologist for positive nuclear staining. RESULTS: No tumors showed immunohistochemical evidence of a type I estrogen receptor. CONCLUSION: Type I estrogen receptors are not present in choroidal melanoma. Estrogens are not likely to influence choroidal melanoma growth through traditional receptors.

Mutants with enhanced nitrogenase activity in hydroponic Azospirillum brasilense-wheat associations.

The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism.

Prognostic value of histologic grade and proliferative activity in axillary node-positive breast cancer: results from the Eastern Cooperative Oncology Group Companion Study, EST 4189.

PURPOSE: The identification of a subset of patients with axillary lymph node-positive breast cancer with an improved prognosis would be clinically useful. We report the prognostic importance of histologic grading and proliferative activity in a cohort of patients with axillary lymph node-positive breast cancer and compare these parameters with other established prognostic factors. PATIENTS AND METHODS: This Eastern Cooperative Oncology Group laboratory companion study (E4189) centered on 560 axillary lymph node-positive patients registered onto one of six eligible clinical protocols. Flow cytometric (ploidy and S-phase fraction [SPF]) and histopathologic analyses (Nottingham Combined Histologic Grade and mitotic index) were performed on paraffin-embedded tissue from 368 patients. RESULTS: Disease recurred in 208 patients; in 161 (77%), within the first 5 years. Mitotic index and grade were associated with both ploidy and SPF (P

Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial.

The safety and immunogenicity of an intramuscular (i.m.) and intradermal (ID) formulation of autoclaved Leishmania (Leishmania) amazonensis vaccine was evaluated in 296 volunteers in a randomized, placebo-controlled, double-blind trial in Colombia. There were 4 vaccination groups: i.m. vaccine, i.m. placebo, ID vaccine, and ID placebo. The ID formulations were mixed with BCG as adjuvant at the time of injection. For each group, 3 vaccinations were given with a 20-day interval between injections, and adverse events were monitored at 20 min, and at 2, 7 and 21 days after each injection. BCG-induced adverse reactions resulted in cancellation of the third vaccine administration in the ID groups. Antibody titres did not differ significantly between the groups. Montenegro skin-test conversion was achieved by 86.4% and 90% of the i.m. vaccine group and by 25% and 5% of the i.m. placebo group 80 days and 1 year after vaccination, respectively. A significant increase in mean Leishmania-antigen lymphocyte proliferation indexes was observed after i.m. vaccine immunization, but not after i.m. placebo immunization, 80 days and 1 year after vaccination. Significant levels of IFN gamma but not IL-10 were observed 1 year after vaccination in the i.m. vaccine group compared to the i.m. placebo group. The good safety profile and evidence of Th1 immune reactions due to i.m. vaccination in this phase-I/II study suggest that a population-based phase-III efficacy trial of the i.m. vaccine should be initiated.

Histologic evaluation of the glenohumeral joint capsule after the laser-assisted capsular shift procedure for glenohumeral instability.

Glenohumeral joint capsule obtained from 42 patients who had undergone an arthroscopic laser-assisted capsular shift procedure was evaluated histologically. A total of 53 samples from the anterior inferior glenohumeral ligament of the joint capsule were collected before and at various times after the procedure (range, 0 to 38 months). Despite glenohumeral instability, joint capsule of the patients before the procedure showed no significant histologic lesions. Laser treatment significantly altered the histologic properties of the tissue as evidenced by hyalinization of collagen and necrotic cells (time 0). Tissues sampled during the short-term period (3 to 6 months) after the procedure demonstrated fibrous connective tissue with reactive cells and vasculature. Collagen and cell morphology returned to normal in the middle- to long-term period (7 to 38 months) after the procedure, while the number of fibroblasts remained elevated. Joint capsule collected from the shoulders of six patients who experienced stiffness after the procedure showed persistent synovial, cellular, and vascular reaction even after 1 year postoperatively, the cause of which is unclear. This study revealed histologic evidence of robust tissue healing and maturation after thermal treatment by the laser-assisted capsular shift procedure, although mechanical and biochemical characterization of the tissue was not evaluated. Correlation with clinical follow-up must be performed to further clarify the advantages and disadvantages of this procedure.

Sonography of testicular tumors and tumor-like conditions: a radiologic-pathologic correlation.

Malignant testicular tumors are an important clinical problem, and ultrasound is the most frequently ordered imaging modality once a palpable scrotal mass is discovered. Numerous articles discussing the role of ultrasound in the evaluation of testicular pathology have confirmed the value of preoperative imaging. This article presents a review of imaging literature regarding testicular neoplasms, with an emphasis on correlation of gross and microscopic tumor pathology and imaging findings. Also included are sections on anatomy, epidemiology, histogenesis, and tumor markers.

Overcoming cellular senescence in human cancer pathogenesis.

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and -3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P = 0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11-q12 (0.99) and +8p22-pter (0.94) in the immortal muscle invasive TCCs, and of -9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13-p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P = 0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P = 0.04) than was p16 or pRb alteration (P = 0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11-q12, -3p13-p14, or -8p21-pter.

Potential use of image analysis for the evaluation of cellular predicting factors for therapeutic response in breast cancers.

OBJECTIVE: To summarize the current literature on the association between biologic prognostic factors and therapeutic implications. STUDY DESIGN: To illustrate how these biologic factors are determined and how they can affect treatment, three patients' biologic profiles and their implications for the patients' choice of therapeutic approaches were analyzed. Immunohistochemical techniques combined with image analysis was used to evaluate estrogen receptors, progesterone receptors, proliferation index and erbB-2. Visual assessment was used to evaluate P glycoprotein (MDR1), EGFR and p53. RESULTS: Data from the literature stress the importance of biologic profiles for defining tumor behavior and patient management. The examples of patients' biologic factors illustrated the possible importance of these factors for helping to design treatment. CONCLUSION: Today the data on the association of patient response to chemotherapy and molecular markers are only starting to accumulate. A larger database is needed for a more precise estimation of response probability in order to help physicians decide between treatment options.

The role of radiotherapy in the management of operable locally advanced breast carcinoma: results of a randomized trial by the Eastern Cooperative Oncology Group.

BACKGROUND: The purpose of this study was to test the role of radiotherapy following total mastectomy, axillary dissection, and adjuvant systemic therapy in the management of operable locally advanced breast carcinoma. METHODS: After undergoing mastectomy and axillary dissection, 426 patients with locally advanced breast carcinoma were registered on study and stratified by patient characteristics and risk factors. All patients were then treated with six courses of chemohormonotherapy. After being restaged, the 332 patients remaining without recurrence were randomized to receive prophylactic radiotherapy or to undergo observation and receive radiotherapy only if and when there was locoregional recurrence. RESULTS: Three hundred twelve of 332 randomized patients were deemed eligible and analyzed for both time to relapse and survival. The median follow-up period was 9.1 years. There were no significant differences in time to relapse and overall survival between the two treatment arms. Of those assigned to radiation, 60% relapsed, with a median time to relapse of 4.7 years, and 46% were alive at last follow-up, with a median survival of 8.3 years. Of those assigned to observation, 56% relapsed, with a median time to relapse of 5.2 years, and 47% were alive at last follow-up, with a median survival of 8.1 years. The two treatment arms had significantly different patterns of sites of first recurrence. There were 9% fewer locoregional first recurrences among those assigned to radiation than among those assigned to observation (15% vs. 24%), whereas there were 15% more first relapses at distant sites (50% vs. 35%) among those assigned to radiation (P = 0.003). CONCLUSIONS: Radiotherapy for locally advanced breast carcinoma, following mastectomy, axillary dissection, and adjuvant systemic therapy, results in fewer locoregional but more distant recurrences at first relapse. No significant advantage was seen for consolidation radiotherapy over observation in terms of either time to relapse or survival, both of which were virtually identical in the two treatment arms. [See editorial counterpoint on pages 1061-6 and reply to counterpoint on pages 1067-8, this issue.]

Heat shock protein 27 overexpression in breast cancer lymph node metastasis.

BACKGROUND: Heat shock protein 27 (hsp-27) is overexpressed in approximately 67% pure ductal carcinoma in situ (DCIS), in approximately 50% DCIS associated with invasive ductal carcinoma (IDC), and in approximately 25% IDC alone. If this decrease in hsp-27 expression has a role in the progression of malignancy in IDC, we postulate a further reduction in expression in nodal metastasis. METHODS: To test this hypothesis, we evaluated the distribution of hsp-27 in primary IDC and in synchronous regional lymph node metastasis within the same patient by immuno-histochemistry. RESULTS: Nine of 30 primary IDCs (30%) and 22 of 30 lymph node metastases (73%) overexpressed hsp-27. Contrary to our hypothesis, of 21 IDCs with no or low hsp-27 expression, 13 (62%) had overexpression of this protein within nodal metastasis. CONCLUSIONS: hsp-27 appears to confer cytoprotection for metastatic cells, which may help explain why hsp-27 overexpression is associated with reduced disease-free survival in breast carcinomas.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Sulcus vocalis: a rational analytical approach to diagnosis and management.

The term sulcus vocalis has been applied to a spectrum of disorders ranging from minor vocal fold indentations to destructive lesions causing severe dysphonia. To clarify the pathophysiology and to develop a more rational approach to treatment, we report a series of sulcus patients including 20 surgical cases. Clinical and histopathologic analysis produced a clinically useful classification: type 1 is a physiologic variant accentuated by atrophy but with intact lamina propria; types 2 (sulcus vergeture) and 3 (sulcus vocalis) are characterized by severe dysphonia, loss of vibratory activity, and destruction of the functional superficial lamina propria. These latter cases respond favorably to microsurgery designed to remove destroyed tissue, release scar contracture, and promote mucosal redraping by regional undermining. Further study of the extracellular matrix of the superficial lamina propria (Reinke's space) might indicate a common pathway in the pathogenesis of sulcus deformities and other related benign vocal fold lesions.

Lack of association between tumor necrosis and hsp-27 expression in primary breast cancer.

Overexpression of heat shock protein 27 (hsp-27) is associated with reduced disease-free survival in early stage breast cancer. Histopathologic evidence of confluent necrosis within primary infiltrating ductal carcinoma (IDC) is similarly an indication of poor prognosis. We postulated that IDC evidencing confluent tumor necrosis (TN) might overexpress this protein, which would help explain why hsp-27 is associated with higher-risk cancers. To test this hypothesis, presence of TN (as opposed to individual cell apoptosis) and of hsp-27 expression by immunohistochemistry were evaluated independently in 48 specimens of IDC. Nineteen (40%) overexpressed hsp-27 and 10 (21%) displayed necrosis. IDCs with areas of TN are less likely to overexpress hsp-27, suggesting a lack of association between these histoprognostic variables. This negative correlation, however, supports hsp-27 as an independent predictor of high-risk disease.

FISH detection of HER-2/neu oncogene amplification in early onset breast cancer.

HER-2/neu (c-erbB-2) gene amplification based on Southern blotting or immunohistochemistry has been shown to be predictive of poor outcome in breast cancer occurring in women over 40, but there is little data on the role of HER-2/neu in young women with breast cancer, many of whom may have inherited BRCA1 or other predisposing genes. The present study used fluorescent in situ hybridization (FISH) on archival specimens of breast cancer from 37 women under the age of 40 to evaluate the role of HER-2/neu amplification in this cohort, and to also evaluate the efficacy of FISH for quantifying amplification. The frequency of primary tumors with a greater than fourfold increase in gene copy number was found to be 38%, which is similar to the frequency of amplification reported in Southern blot studies in older women. However, the greater sensitivity of FISH enabled detection of low level amplification (more than 2 but less than 8 gene copies), which was found in an additional 30% of the tumors. Patients with low level amplification demonstrated a 54% recurrence rate, compared to 86% in those with high amplification and 17% in those with no amplification. HER-2/neu amplification appeared to be more prognostic of recurrence than nodal status, with 45% of node negative tumors recurring compared to 62% of those which were node positive, nor was tumor size predictive of recurrence in this cohort since tumors of 2 cm or less recurred in 44% of cases compared to 57% of those larger than 2 cm. Thus, this study demonstrates that FISH is a reproducible and sensitive technique for detecting HER-2/neu amplification, and that amplification of the oncogene is the strongest independent indicator of recurrence of breast cancer in young women.

Autoimmune diabetes: caught in the causality trap?

The lack of concordance between genotype and clinical diabetes has prompted a search for the infectious agent that precipitates this autoimmune disease. However, this approach may be misleading. It assumes that the disease-prone individuals that do not develop diabetes do not have autoimmunity. In the non-obese diabetic (NOD) mouse, the genotype is a primary determinant of autoimmunity. Not all animals of the disease-prone genotype develop clinical disease; however, all have autoimmunity. This is expressed as a destructive or non-destructive process. Multiple pathways are open to the immune system and whether or not the immune response is destructive and leads to the development of clinical disease, appears to be a random process. If this is the case, the most important questions relating to autoimmune disease are not those concerning the 'causative' agents. Instead we should be asking what are the differences between pathways open to the immune system and what factors affect the probability that one or another pathway is finally selected?

Abnormal rectal mucosa of the anal transitional zone in ulcerative colitis.

OBJECTIVE: To determine the frequency of atypia and active ulcerative colitis (UC) in rectal mucosa within the anal transitional zone (ATZ). DESIGN: Surgeons identified ATZ tissues from restorative proctocolectomy specimens for determination by surgical pathologists of specific histopathologic features in rectal mucosa of the ATZ. SETTING: Surgical referral center for restorative proctocolectomy. PATIENTS: Ninety-four patients with symptomatic UC underwent restorative proctocolectomy between January 1991 and December 1994. INTERVENTIONS: Specific histopathologic features of active UC in the ATZ were evaluated by a single reviewer who did not know the clinicopathologic details of individual study patients. MAIN OUTCOME MEASUREMENTS: Presence and coexistence of rectal mucosal dysplasia (high or low grade), mucosa classified as indefinite for dysplasia, and acute UC (crypt abscess or cryptitis) in the ATZ. RESULTS: Of 94 ATZ tissue specimens, acute intracryptic inflammation was present in 60 rectal mucosa specimens (64%). In 29 (48%) of these 60 specimens, inflammation was neither widespread nor intense. Rectal mucosal dysplasia (low grade but not high grade) was present in 15 (16%) of 94 ATZs specimens. Inflammation elsewhere in the rectal mucosa accompanied dysplasia in 11 (73%) of 15 ATZ specimens. Rectal mucosa classified as indefinite for dysplasia was present in 24 (26%) of 94 ATZ specimens and coexisted with inflammation in 15 (63%) of these 24. Thus, rectal mucosal atypia was present in 39 (41%) of 94 ATZ specimens, and in 26 (67%) of these 39, abnormal rectal mucosa coexisted with acute inflammation. CONCLUSIONS: Rectal mucosa in the ATZ can exhibit active UC and/or atypia. Long-term monitoring is advisable if the ATZ is preserved during restorative proctocolectomy.