Rajni Chibbar

Reduced PTEN expression predicts relapse in patients with breast carcinoma treated by tamoxifen

Tamoxifen treatment substantially improves the 10-year survival of women with estrogen-receptor (ER)-α-positive tumors. However, approximately one-third of all breast cancer patients with ER-α-positive tumors progress on antiestrogen therapy. The molecular mechanism(s) involved in antiestrogen-resistant phenotype of breast carcinoma is not completely understood. The PTEN (phosphatase and tensin homolog deleted on chromosome Ten) gene is a novel candidate tumor suppressor that plays an important role in cell cycle regulation and apoptosis by regulating Protein kinase-B/Akt activity. Previous studies have shown that PTEN downregulation in breast cancer is associated with high-grade tumor, distant metastases and poorer disease-free survival. Decreased PTEN and/or increased protein kinase B/Akt activity in breast cancer cells has recently been associated with resistance to tamoxifen-induced apoptosis. In this study, we have evaluated PTEN expression by immunohistochemistry in 100 tamoxifen-treated ER-α-positive breast cancer patients. Reduced PTEN protein expression was associated with shorter relapse-free survival. When stage I patients were analyzed separately, reduced PTEN expression was a strong predictor of both, shorter relapse-free survival and shorter disease-specific survival. An association of reduced PTEN expression with shorter relapse-free survival and disease-specific survival in stage I patients was still observed after stratification by stage, axillary lymph node status, tumor size, grade, and expression of ER-α, progesterone receptor, and Her-2/neu. In summary, our results showed a strong association between downregulation of PTEN expression in ER-α-positive tumors and failure to tamoxifen treatment.

Nonspecific interstitial pneumonia and usual interstitial pneumonia with mutation in surfactant protein C in familial pulmonary fibrosis

Nonspecific interstitial pneumonia, a recently described form of idiopathic interstitial pneumonia, is characterized by uniform involvement of the alveolar septae with interstitial inflammation and variable amounts of fibrosis. Histological observations differentiate nonspecific interstitial pneumonia from usual interstitial pneumonia and clinically, patients with a nonspecific interstitial pneumonia pattern show better prognosis than those with usual interstitial pneumonia. We have genetically analyzed a family with a history of usual interstitial pneumonia. Most of the patients presented as adults and their biopsies showed a pattern consistent with usual interstitial pneumonia. However, three family members presented in early childhood and their biopsies revealed a nonspecific interstitial pneumonia pattern. The inheritance pattern of usual interstitial pneumonia is consistent with autosomal dominant inheritance with variable expression. DNA sequence analyses of the surfactant protein C gene in children with nonspecific interstitial pneumonia and adults with usual interstitial pneumonia exhibit a common heterozygous mutation located in exon 5. The mutation causes a Leu188 to Gln188 change in the carboxy-terminal region of prosurfactant protein C, possibly affecting peptide processing. These observations suggest that individuals with this particular mutation in surfactant protein C gene might be at increased risk of interstitial lung disease of variety of types.

Dietary soy isoflavones increase insulin secretion and prevent the development of diabetic cataracts in streptozotocin-induced diabetic rats

Soy isoflavone-containing diets have been reported to be beneficial in diabetes. This present study investigated the hypoglycemic effects of isoflavones in streptozotocin (STZ)-induced diabetes. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of 100 mg/kg STZ. Diabetic rats were then randomly divided into 3 groups and received a special diet supplemented with casein (control), low-isoflavone soy (LIS) protein, and high-isoflavone soy protein (HIS) for 8 weeks. Compared with the control or LIS groups, those rats on the HIS diet had significantly increased body weight and serum insulin levels and reduced serum glucose and methylglyoxal levels. Serum glutathione levels were also increased in rats given the HIS diet compared with those in the control or LIS (P < .01). Serum high-density lipoprotein cholesterol level was significantly higher in HIS-fed rats than that of the control or LIS rats (P < .05). More importantly, the death rate and incidence of cataracts in the diabetic rats were markedly decreased in the HIS group. In conclusion, ingestion of high-isoflavone soy protein not only lowers glucose levels but also reduces the incidence of cataracts in diabetic rats. The beneficial effects of soy isoflavones are attributed to increased insulin secretion, a better glycemic control, and antioxidant protection.

Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8 CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558HS expressing cytoplasmic HSP70, and purified EXOHSP and EXOHS from J558HSP and J558HS tumour cell culture supernatants by ultracentrifugation. We found that EXOHSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Iab, CD40, CD80 and inflammatory cytokines than EXOHS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 106 cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXOHSP are able to stimulate type 1 CD4+ helper T (Th1) cell responses, and more efficient P1A‐specific CD8+ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXOHS. In addition, we further elucidate that EXOHSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8+ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.

Dendritic Cells Recruit T Cell Exosomes via Exosomal LFA-1 Leading to Inhibition of CD8+ CTL Responses through Downregulation of Peptide/MHC Class I and Fas Ligand-Mediated Cytotoxicity

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8+ T cells by cultivation of OVA-pulsed dendritic cells (DCOVA) with naive CD8+ T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8+ T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DCOVA-mediated CD8+ CTL responses and antitumor immunity. We found that DCOVA uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I–TCR and CD54–LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DCOVA via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8+ T cell EXOs, can inhibit DCOVA-stimulated CD8+ CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DCOVA-mediated CD8+ CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti–LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8+ CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8+ TCRs have a similar inhibitory effect as T cell EXOs in DCOVA-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8+ T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

Tumor-associated antigen 90K/Mac-2-binding protein : Possible role in colon cancer

The tumor‐associated antigen 90K (TAA90K)/Mac‐2‐binding protein implicated in cancer progression and metastasis is modified by β1‐6 branched N‐linked oligosaccharides in colon cancer cells, glycans shown to contribute to cancer metastasis. To elucidate the role of TAA90K in colon cancer, we examined its expression and function in human colon tumors and colon carcinoma cell lines. Immunohistochemical analyses of colon tumors revealed elevated expression of TAA90K in all samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we carried out protein and cell binding assays using TAA90K‐His purified from HT‐29 cells colon carcinoma cells infected with recombinant vaccinia virus expressing TAA90K containing a C‐terminal poly‐histidine tag. TAA90K‐His bound to fibronectin, collagen IV, laminins‐1, ‐5, and ‐10 and galectin‐3 (Mac‐2) but poorly to collagen I and galectin‐1. As expected, binding of TAA90K to galectin‐3 was dependent on carbohydrate since it was inhibitable by lactose and asiolofetuin, and a TAA90K‐His glycoform purified from HT‐29 cells treated with the glycosylation inhibitor 1‐deoxymannojirimycin bound poorly to galectin‐3. Unlike TAA90K isolated from other cell types, TAA90K‐His isolated from colon cancer cells failed to mediate adhesion of colon cancer and normal cell lines, possibly due to cell‐type specific glycosylation of TAA90K‐His and/or its putative cellular receptor. However, at low concentrations, TAA90K‐His enhanced galectin‐3‐mediated HT‐29 cell adhesion while at high concentrations, it inhibited cell adhesion. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins, including galectin‐3. J. Cell. Biochem. 98: 1351–1366, 2006.

Tumor Apoptotic Bodies Inhibit CTL Responses and Antitumor Immunity via Membrane-Bound Transforming Growth Factor-β1 Inducing CD8+ T-Cell Anergy and CD4+ Tr1 Cell Responses

Tumor cell apoptosis induced by radiation therapy results in apoptotic tumor cells and apparition of membrane blebs termed apoptotic bodies (APB). The immune responses induced by apoptotic tumor cells have been extensively studied. However, the role of APB in modulation of tumor immune responses is elusive. In this study, we induced apoptosis in 90% ovabumin-expressing EG7 tumor cells by in vitro irradiation (9,000 rad) of tumor cells with a subsequent cell culture for 9 hours. APB purified from irradiation-induced apoptotic EG7 cell culture supernatant by differential ultracentrifugation were vesicles with 50 to 90 nm in diameter and expressed apoptosis-specific Annexin V, 14-3-3, and Histone H3. We then investigated its potential modulation in DC(OVA)-induced T-cell responses and antitumor immunity. We found that EG7-derived APB were tolerogenic and capable of suppressing DC(OVA)-stimulated CD8+ CTL responses and antitumor immunity via its induction of CD8+ T-cell anergy and type 1 regulatory CD4+ T-cell responses. Analysis of apoptotic tumor cells and APB revealed the expression of membrane-bound transforming growth factor (TGF)-beta1 associated with irradiation-induced apoptosis formation, which is a result from activation of transcriptional factor NF-AT specific for TGF-beta1 promoters. Our data further elucidate that it is the membrane-bound TGF-beta1 expression on APB that contributes to its in vitro antiproliferative effect as shown by using neutralizing TGF-beta1-specific antibody. Administration of anti-TGF-beta1 antibody in vivo also blocked APB-mediated immune suppression of CD8+ CTL responses and antitumor immunity. Therefore, our study may have great impact in designing a combined radiation therapy with immunotherapy of cancer.

Synovial Chondromatosis and Chondrosarcoma: A Diagnostic Dilemma

Purpose: The progression of synovial chondromatosis to chondrosarcoma is very rare. Distinction between these two entities may be difficult on histology alone, and should be based on clinical, radiographic and microscopic evidence. Immunohistochemical markers that would facilitate differentiation between synovial chondromatosis and chondrosarcoma are currently being investigated. Patients: We describe the cases of two patients who presented with synovial chondromatosis and progression to synovial chondrosarcoma during periods of 7 and 11 years. Several biopsies and resected specimens demonstrated synovial chondromatosis before a diagnosis of chondrosarcoma was made. Method: We have examined five markers (Bcl2, Ki67, p27, p16, and p53) in all specimens from these cases, as well as known cases of chondromatosis and chondrosarcoma for control purposes. Results: We found increased expression of Bcl2 in benign chondromatosis compared to synovial or central chondrosarcomas. Discussion: Distinction between chondromatosis and its progression to low grade chondrosarcoma is difficult at histological level, and must involve incorporation of clinical and radiographical data. Although preliminary, our study suggests that reduced or absent expression of Bcl2 is associated withmalignant transformation of chondromatosis.

GP120-specific exosome-targeted T cell-based vaccine capable of stimulating DC- and CD4(+) T-independent CTL responses.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics. In this study, we generated ovalbumin (OVA)-pulsed and pcDNAgp120-transfected dendritic cell (DC)-released exosomes (EXOova and EXOgp120) and ConA-stimulated C57BL/6 CD8(+) T cells. OVA- and Gp120-Texo vaccines were generated from CD8(+) T cells with uptake of EXOova and EXOgp120, respectively. We demonstrate that OVA-Texo stimulates in vitro and in vivo OVA-specific CD4(+) and CD8(+) cytotoxic T lymphocyte (CTL) responses leading to long-term immunity against OVA-expressing BL6-10(OVA) melanoma. Interestingly, CD8(+) T cell responses are DC and CD4(+) T cell independent. Importantly, Gp120-Texo also stimulates Gp120-specific CTL responses and long-term immunity against Gp120-expressing B16 melanoma. Therefore, this novel HIV-1-specific EXO-targeted Gp120-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients with DC dysfunction and CD4(+) T cell deficiency.

An Expert Support System for Breast Cancer Diagnosis using Color Wavelet Features

Breast cancer diagnosis can be done through the pathologic assessments of breast tissue samples such as core needle biopsy technique. The result of analysis on this sample by pathologist is crucial for breast cancer patient. In this paper, nucleus of tissue samples are investigated after decomposition by means of the Log-Gabor wavelet on HSV color domain and an algorithm is developed to compute the color wavelet features. These features are used for breast cancer diagnosis using Support Vector Machine (SVM) classifier algorithm. The ability of properly trained SVM is to correctly classify patterns and make them particularly suitable for use in an expert system that aids in the diagnosis of cancer tissue samples. The results are compared with other multivariate classifiers such as Naïves Bayes classifier and Artificial Neural Network. The overall accuracy of the proposed method using SVM classifier will be further useful for automation in cancer diagnosis.