Expression of Wilms tumor gene (WT1) in epithelial ovarian cancer

OBJECTIVES: The identification of proteins that are selectively expressed in cancer and with potential to elicit an immune response is the first step towards antigen-specific immunotherapy. The Wilms tumor gene product (WT1) is inherently immunogenic and is now thought to be oncogenic. The aim of this study was to determine the expression of WT1 in epithelial ovarian cancer (EOC) and correlate with clinico-pathologic characteristics. METHODS: WT1 expression was examined using immunohistochemistry applied on a tissue microarray of normal tissues and a panel of 100 EOC tissues. The distribution of WT1 expression and clinico-pathologic variables were analyzed. Survival probabilities were estimated by Kaplan-Meier method, and statistical significance was determined by the log-rank test. RESULTS: WT1 expression was observed in 78/100 of specimens. The predominant expression pattern was homogenous, occurring in 66/100 (66%) of WT1-positive specimens, while 12/100 (12%) demonstrated heterogeneous staining. In normal tissues, WT1 expression was noted in kidneys, splenic capsule, Sertoli cells of the testis, and granulosa cells of the ovary. The median follow-up of the patient population was 30 months. Patients with WT1-positive tumors tended to have a higher grade (P = 0.006) and stage (P = 0.002) of tumor. However, there were no significant differences in the distribution of patients with WT1-positive tumors in relation to disease-free and overall survival. CONCLUSIONS: Our data demonstrate that WT1 is expressed at high frequency in patients with EOC. Since WT1 demonstrates tissue-restricted expression and is inherently immunogenic, it could represent an attractive target for antigen-specific immunotherapy in EOC.     

Polarized expression of immunoglobulin, spectrin, and protein kinase C beta II occurs in B cells from normal BALB/c, autoimmune lpr, and anti-ssDNA transgenic, tolerant mice.

The rapid redistribution of B cell surface immunoglobulin to a cap upon cross-linking treatment is a well-described phenomenon, the physiological significance of which is unknown. We describe the observation that splenic B cells from unimmunized normal, autoimmune, and tolerant mice express naturally occurring capped immunoglobulin in the absence of exogenous stimulation. The percentage of capped B cells increases to 20% of B cells by age 16 weeks in the progressive autoimmune lpr mouse. Transgenic, tolerant mice expressing lpr-derived genes for ssDNA-binding antibody also demonstrate a large percentage (35-75%) of immunoglobulin-capped splenic B cells. In these capped B cells, protein kinase C beta II, the cytoskeletal proteins spectrin and ankyrin, and the lipophilic probe diI are enriched beneath the site of the immunoglobulin cap. These data suggest that polarization of surface receptors, signaling molecules, anionic phospholipid domains, and cytoskeletal proteins may be an important part of the B cell immune response in vivo.     

偏盲患者在听-视觉刺激后视觉搜索能力的改善

目的：识别癌组织中选择性表达和可能引起免疫反应的蛋白，是抗原特异性免疫疗法的第一步。Wilms肿瘤基因（WT1）产物具有天生的免疫原性，现认为其具致癌性。本研究目的是测定WT1在卵巢上皮癌（EOC）中的表达，并了解其与临床及病理学特点的关系。方法：用微阵列免疫组化法检测WT1的表达，检测对象为正常组织和100个EOC组织。分析WT1表达的分布情况及临床-病理学变量。生存概率使用Kaplan-Meier方法评估，统计方法为秩和检验。结果：100例中，78例发现有WT1表达，其中66例（66％）为同质性染色，为主要表达类型，12例（12％）为异质性染色。     

Chaperoning function of stress protein grp170, a member of the hsp70 superfamily, is responsible for its immunoadjuvant activity

When used as vaccines, tumor-derived stress proteins can elicit antitumor immune responses. For members of the hsp70 superfamily, like grp170, this seems to be due to (a) the chaperoning of antigenic peptide by the stress protein and (b) the binding of the stress protein to receptor(s) on antigen-presenting cells (APC) and subsequent antigen presentation. This suggests that domains exist on the stress protein for each function. In this study, we determine the ability of grp170 and its structural domains to (a) bind to and present melanoma-associated antigen gp100 to the immune system and (b) to bind to receptors on APCs. A direct correlation between chaperone function, binding to APCs in a receptor-like manner, and antitumor immunity was observed. Two mutants that share no common sequence, yet are both effective in their antitumor activities, compete with one another for APC binding. Studies of other members of the hsp70 superfamily, hsp110 and hsp70, or their domain deletion mutants, further confirmed that APC binding segregates with chaperoning function and not sequence. Therefore, these studies suggest that molecular chaperoning is involved in stress protein interactions with APCs, antigen binding, and in eliciting antitumor immunity, thus bridging this ancient function of stress proteins in prokaryotes to their ability to elicit immunity in higher organisms.     

Low-level laser effect on neurosensory recovery after sagittal ramus osteotomy

OBJECTIVES: This study examined the potential benefit of perioperative and short-term postoperative low-level laser (LLL) therapy on objective and subjective neurosensory recovery after bilateral sagittal split osteotomy surgery. METHODS: Six consecutive patients undergoing bilateral sagittal split osteotomy procedures were enrolled in this prospective study. A complete preoperative clinical neurosensory test, consisting of brush stroke directional discrimination, 2-point discrimination, contact detection, pin prick nociception, and thermal discrimination, was performed on each patient; and a subjective assessment of neurosensory function was made by using a visual analog scale (VAS). The protocol for LLL treatments consisted of real LLL (4 x 6 J per treatment) along the distribution of the inferior alveolar nerve at 4 sites, for a total of 7 treatments, delivered immediately before surgery; at 6 and 24 hours after surgery; and on postoperative days 2, 3, 4, and 7. The clinical neurosensory test and VAS were completed just before each of the treatment sessions and on days 14 and 28, by one examiner. RESULTS: When the results of the patients treated with LLL were compared with published values for neurosensory recovery after orthognathic surgery, there was a significant acceleration in the time course, as well as in the magnitude, of neurosensory return. Brush stroke directional discrimination approached normal values by 14 days, whereas 2-point discrimination and contact detection showed significant improvement at 14 days and returned to near-normal values by 2 months. The results of thermal discrimination and pin prick nociception revealed few neurosensory deficits; however, those patients who were affected showed a slower recovery trend and remained neurosensory-deficient for up to 2 months. The VAS analysis revealed a rapidly progressive improvement in subjective assessment, showing a 50% deficit at 2 days and only a 15% subjective deficit at 2 months. CONCLUSIONS: This study demonstrates that neurosensory recovery after bilateral sagittal split osteotomy procedures can be significantly improved, both in terms of time course and magnitude of return of function, with the adjunctive use of LLL therapy.     

Increased concentration of spectrin is observed in avian dystrophic muscle.

A significant increase in the concentration of spectrin has been observed in dystrophic chicken pectoralis major muscle when compared to normal fast-twitch muscle. In normal muscle, alpha-spectrin-specific immunofluorescence delineates each myofiber with a network pattern of staining at the sarcolemma with little staining within the cytoplasm. In dystrophic fibers, numerous intensely stained areas occur within the cytoplasm and staining at the sarcolemma is increased, thereby obscuring or eliminating the highly regular network arrangement of spectrin usually seen in this region. When immunofluorescence experiments are performed on microsomal vesicles isolated from normal and dystrophic tissues, only a small fraction of normal vesicles are stained, whereas most of the dystrophic vesicles are associated with spectrin. An increase in spectrin concentration is observed using immunoautoradiography of whole muscle and isolated microsomes, thus supporting the immunofluorescent observations described above. The early-age post-hatching when increases in spectrin concentration can be detected and the simplicity of the immunofluorescent technique make this observation useful as a new diagnostic parameter. This observation also shows that the distribution of spectrin and its concentration within nonerythroid cells can be modified by abnormal physiological states; this modification may contribute to subsequent symptoms, such as increased rigidity and abnormal calcium metabolism, that are observed in dystrophy.     

Mutation rate of MAP2K4/MKK4 in breast carcinoma

The stress‐activated protein kinase (SAPK) pathways represent phosphorylation cascades that convey pro‐apoptotic signals. The relevant inputs include Ras proteins as well as exposure of cells to ultraviolet light, tumor‐necrosis factor, and other stress‐related inputs. The mitogen‐activated protein kinase kinase (MAPKK) homolog MAP2K4 (MKK4, SEK, JNKK1) is a centrally‐placed mediator of the SAPK pathways. MAP2K4 mutations or homozygous deletions are reported in about 5% of a wide variety of tumor types. The exception is breast cancer, where genetic inactivation in 3 of 22 (15%) cell lines had suggested that the mutational involvement of MAP2K4 might be accentuated in this tumor type. This finding might have represented an important difference, or solely a chance numerical variation. To address this question, we studied an independent panel of 20 breast cancer cell lines and xenografts for MAP2K4 alterations. We found a splice acceptor mutation accompanied by loss of the other allele in the cell line MPE600. This was the sole alteration in this panel (5% of tumors). These data seem to re‐establish a rather consistent rate of genetic inactivation of MAP2K4 among most tumor types, including breast cancer. The genetic evaluation of other mediators of the SAPK pathways might offer insight into a promising, but as yet poorly defined, tumor‐suppressive system. © 2001 Wiley‐Liss, Inc.     

Comparison of the effects of two different whole body hyperthermia protocols on the distribution of murine leukocyte populations.

Two predominant WBH protocols presently being used in clinical trials include a low temperature, long duration (LL) WBH, where core body temperature is raised to 39.5-40 degrees C for 6h or more, and a high temperature, short duration (HS) WBH, where core body temperature is raised to 41.8 degrees C for up to 2h. Here, the effects of LL-WBH and HS-WBH on leukocyte populations in the blood, spleen, lymph node (LN) and peritoneal cavity (PerC) of Balb/c mice were compared using flow cytometry. The total numbers of peripheral blood leukocytes decreased up to 2-fold immediately after LL-WBH, reflecting a decrease of lymphocyte numbers compared to controls. In contrast, the numbers of blood leukocytes are increased 2.7-fold immediately after HS-WBH compared to controls, reflecting an increase in lymphocytes, monocytes and granulocytes. After both LL- and HS-WBH treatment, leukocyte numbers in the spleen are decreased approximately 2-fold, again reflecting decreases in lymphocyte numbers. In the PerC, total numbers of leukocytes are also significantly decreased (2-fold) during LL-WBH but not HS-WBH. Total numbers of leukocytes in the LNs were unaffected by both LL- and HS-WBH. Overall, these data reveal differential effects of the LL- and HS-WBH protocols on leukocyte populations in the blood, spleen, LN and PerC of Balb/c mice.     

IL-6 trans-signaling licenses mouse and human tumor microvascular gateways for trafficking of cytotoxic T cells

Immune cells are key regulators of neoplastic progression, which is often mediated through their release of cytokines. Inflammatory cytokines such as IL-6 exert tumor-promoting activities by driving growth and survival of neoplastic cells. However, whether these cytokines also have a role in recruiting mediators of adaptive anticancer immunity has not been investigated. Here, we report that homeostatic trafficking of tumor-reactive CD8+ T cells across microvascular checkpoints is limited in tumors despite the presence of inflammatory cytokines. Intravital imaging in tumor-bearing mice revealed that systemic thermal therapy (core temperature elevated to 39.5°C ± 0.5°C for 6 hours) activated an IL-6 trans-signaling program in the tumor blood vessels that modified the vasculature such that it could support enhanced trafficking of CD8+ effector/memory T cells (Tems) into tumors. A concomitant decrease in tumor infiltration by Tregs during systemic thermal therapy resulted in substantial enhancement of Tem/Treg ratios. Mechanistically, IL-6 produced by nonhematopoietic stromal cells acted cooperatively with soluble IL-6 receptor-α and thermally induced gp130 to promote E/P-selectin- and ICAM-1-dependent extravasation of cytotoxic T cells in tumors. Parallel increases in vascular adhesion were induced by IL-6/soluble IL-6 receptor-α fusion protein in mouse tumors and patient tumor explants. Finally, a causal link was established between IL-6-dependent licensing of tumor vessels for Tem trafficking and apoptosis of tumor targets. These findings suggest that the unique IL-6-rich tumor microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy.