Anurag Saxena

Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells

Dendritic cells (DCs) that acquired antigen from apoptotic tumor cells are able to induce major histocompatibility complex (MHC) class I‐restricted cytotoxic T lymphocytes and antitumor immunity. In the present study, we investigated the efficiency of antitumor immunity derived from DCs that had phagocytosed apoptotic/necrotic BL6‐10 melanoma cells compared with that of DCs pulsed with the tumor mTRP2 peptide. Our data showed that phagocytosis of apoptotic/necrotic tumor cells resulted in maturation of DCs with up‐regulated expression of proinflammatory cytokines [interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α, interferon‐γ and granulocyte‐macrophage colony‐stimulating factor], chemokines (MIP‐1α, MIP‐1β and MIP‐2), the CC chemokine receptor CCR7 and the cell surface molecules (MHC class II, CD11b, CD40 and CD86), and down‐regulated expression of the CC chemokine receptors CCR2 and CCR5. These mature DCs displayed enhanced migration toward the CC chemokine MIP‐3β in a chemotaxis assay in vitro and to the regional lymph nodes in an animal model in vivo. Our data also showed that vaccination with DCs that had phagocytosed apoptotic/necrotic BL6‐10 cells was able to (i) more strongly stimulate allogeneic T‐cell proliferation in vitro, (ii) induce an in vivo Th1‐type immune response leading to more efficient tumor‐specific cytotoxic CD8+ T‐cell‐mediated immunity and (iii) eradicate lung metastases in all 6 vaccinated mice compared with mice vaccinated with DCs pulsed with the tumor mTRP2 peptide, in which lung metastases were reduced (mean number of 16 per mouse) but not completely eradicated. Therefore, DCs that had phagocytosed apoptotic/necrotic tumor cells appear to offer new strategies in DC cancer vaccines.

Neutrophil-mediated acute lung injury after extracorporeal perfusion

A pulmonary injury of varying severity occurs routinely after cardiopulmonary bypass. We studied the pulmonary complications of partial cardiopulmonary bypass in four groups of dogs to better define the injury and to evaluate the efficacy of two interventions (addition of a leukocyte filter or cyclooxygenase inhibition) on preservation of systemic oxygenation. All animals received a standard anesthetic (pentobarbital, morphine, and vecuronium) and, after sternotomy, three groups of animals received 3 hours of partial cardiopulmonary bypass. The animals were randomized to receive partial bypass alone (n = 6), indomethacin and bypass (n = 5), or a leukocyte filter and bypass (n = 5). A fourth group (n = 5) did not receive bypass and served as a time control. We measured blood gases and also obtained histologic samples to assess the degree of lung injury. We found that bypass alone caused a significant reduction (p < 0.05) in arterial oxygen tension 1 hour after the conclusion of bypass (175 +/- 53 mm Hg) compared with prebypass values (357 +/- 41 mm Hg). Pretreatment with indomethacin ameliorated the decrease in arterial oxygen tension from prebypass to postbypass values (477 +/- 50 mm Hg versus 339 +/- 57 mm Hg, respectively). Similarly use of a leukocyte filter reduced the decline in arterial oxygen tension from prebypass to postbypass values (440 +/- 71 mm Hg versus 311 +/- 73 mm Hg, respectively). We believe that indomethacin ameliorates the decline in systemic oxygenation associated with bypass by augmentation of hypoxic pulmonary vasoconstriction and that the leukocyte filter acted to reduce pulmonary edema and thereby minimized intrapulmonary shunt.

Goblet Cell Carcinoids of the Appendix Immunophenotype and Ultrastructural Study

BACKGROUND Goblet cell carcinoids of the appendix are rare neoplasms with uncertain biological behavior. OBJECTIVE The aims of our study were to evaluate the immunophenotype of this neoplasm with cell cycle/cell proliferation markers and to understand their histogenesis with ultrastructural analysis using conventional carcinoids as a frame of reference. METHODS Clinical data and archival pathologic material of all goblet cell carcinoids of the appendix recorded by the Saskatchewan Cancer Registry between 1970 and 1998 were reviewed and evaluated by light microscopy, histochemistry, immunohistochemistry, and electron microscopy. RESULTS Seven cases of goblet cell carcinoids were identified among 110 cases of conventional carcinoids of the appendix. Histopathology revealed widespread infiltration of the periappendiceal fat in all cases, with extensive perineural invasion. The cells stained strongly positive for mucicarmine, periodic acid-Schiff, periodic acid-Schiff diastase, Alcian blue, cytokeratin, and carcinoembryonic antigen. Most cases were positive for synaptophysin. Increased expression of cell proliferation markers and cell cycle markers was observed. Expression of p53 was strong in one case. Electron microscopy demonstrated the presence of mucinous vacuoles of varying sizes and occasional membrane-bound neuroendocrine granules. CONCLUSIONS Goblet cell carcinoids of the appendix arise from a pluripotent cell with divergent neuroendocrine and mucinous differentiation. These neoplasms are widely invasive; they demonstrate a high cellular proliferation rate and dysregulation of the cell cycle with up-regulation of cyclin D1 and p21, and down-regulation of p16. Complete removal of the tumor is recommended because of the unpredictable biological behavior of this tumor, which includes delayed local recurrences and lung metastases.

Clonal B cell populations in a minority of patients with Hashimoto's thyroiditis

Background: Hashimoto’s thyroiditis (HT) is a risk factor for thyroid lymphoma, and clonal B cell populations in HT support this link. The literature on B cell clonality in HT is controversial. Aims: To identify clonal B cell populations in HT and to assess their usefulness in differentiating HT from mucosa associated lymphoid tissue (MALT) lymphoma and predicting future development of lymphoma. Methods: DNA from formalin fixed, paraffin wax embedded blocks of thyroid specimens from 10 patients with HT and two thyroid MALT lymphomas was analysed for B cell clonality by seminested polymerase chain reaction (PCR) using FRIII/LJH and FRIII/VLJH primers to amplify the IgH gene VDJ region. In one case, PCR products were sequenced. Immunohistochemistry was performed by labelled streptavidin–biotin technique using antibodies to: CD45, CD45RO, CD3, CD20, and cytokeratin. Results: The histopathological and clinical findings were characteristic of HT. Clonal bands were seen in three and a polyclonal smear pattern was seen in seven cases. The clonal bands in HT were associated with a background smear, and could not be reproduced from other blocks from the same case or from deeper sections of the same block. The clonal bands in thyroid lymphomas were not associated with a background smear and were reproducible. None of the patients with clonal B cells has developed malignant lymphoma during a follow up of 10–13 years. Conclusions: B cell clonal bands in HT have different features from those in lymphoma (non-pure and non-reproducible) and do not predict future development of lymphoma.

Clonal relationship between Hashimoto thyroiditis and thyroid lymphoma

Background: Although Hashimoto thyroiditis (HT) is a predisposing factor for B-lineage thyroid lymphoma, clonal B-cell populations in HT are rare. Aim: To investigate whether there is a clonal relationship between HT and primary thyroid lymphoma. Methods: Clonalilty and sequence similarity was determined by PCR followed by sequencing and comparing immunoglobulin heavy chain (IgH) gene rearrangement sequences to germline sequences and to each other. Results: 12/20 patients with primary thyroid lymphoma had a previous history and histological diagnosis of HT. Clonal IgH bands associated with a polyclonal background were present in four of these 12 cases of HT; of these four, three had reproducible clonal IgH bands from the subsequently developed lymphoma. The range of similarity (homology) of multiple clonal bands in HT with the germline IgH varied from 90% to 96.3%. Multiple clonal bands in HT had sequence similarity (homology) of 62–100% with the clonal band in the lymphoma from the same patient. At least one clonal band in HT had more than 96% similarity (homology) with the clonal band of lymphoma in all three cases. Conclusion: Sequence similarity between the clonal bands in HT and subsequently developed thyroid lymphoma is supportive of the argument that primary thyroid lymphoma may evolve from HT.

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC(IMAT)) or relative mature DCs (DC(RMAT)). DC(RMAT) displayed an up-regulated expression of immune molecules (Ia(d), CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC(IMAT). These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC(RMAT)/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1x10(6) cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC(IMAT)/J558 hybrids. Furthermore, immunization of mice with engineered DC(RMAT)/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC(RMAT)/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

Biologic and clinical significance of CD7 expression in acute myeloid leukemia.

CD7 antigen, a T‐cell lineage associated antigen, is expressed in a minority of patients with acute myeloid leukemia (AML). The biologic and clinical significance of this finding is not clearly established. In this retrospective study of patients with de novo acute myeloid leukemia, we have identified CD7 expression and analyzed its association with markers expressed early in hemopoietic ontogeny and clinical parameters. Among 60 consecutive AML patients, we found six (10%) expressing CD7 on leukemic cells. There were five males and one female and the mean age was 59.6 years (age range: 32–76 years) with no demographic peculiarities. The FAB subtypes were: M0 (2), M1 (1), M2 (1), and M4 (2). CD7 expression was associated with immature antigens CD34, HLA‐DR, and terminal deoxynucleotidyl transferase (TdT) and antigen receptor gene rearrangements (rearrangements of T‐cell receptor gamma chain in 6/6 and immunoglobulin heavy chain in 2/6). Hepatomegaly was present in three and this was associated with splenomegaly with lymphadenopathy in one patient. Mediastinal or central nervous system involvement was absent. Complete remission was achieved in two patients with standard chemotherapy; one of these is in remission and alive (5 years later), while one died following relapse 9 months later. Three patients had significantly lower response to standard therapeutic regimen (two died during induction and one died 7 months later without ever achieving complete remission). One patient has been excluded in determining the prognostic significance of CD7 due to early death. Our results suggest origin of CD7+ AML from early hemopoietic precursors and indicate biologic aggressiveness in a significant proportion of patients. We suggest evaluation of CD7 in all patients with AML at the time of diagnosis in view of poor clinical outcome. Am. J. Hematol. 58:278–284, 1998.

Expression of Bcl-x, Bcl-2, Bax, and Bak in endarterectomy and atherectomy specimens

The regulation of apoptosis in atherosclerosis is not completely defined. The aim of this study was to determine the expression of Bcl‐2, Bcl‐x, Bax, and Bak in relation to apoptosis in advanced atherosclerotic lesions. In atherectomy (15), endarterectomy (10), and control non‐atherosclerotic segments of renal (2) and of coronary and carotid (5) arteries, the extent of apoptosis was determined using TdT dUTP nick end labelling (TUNEL) and nuclear morphology (karyorrhexis/pyknosis) and expression of apoptosis regulators by immunohistochemistry and western blot analysis on paraffin‐embedded material. In all specimens, the atherosclerotic involvement was advanced: grade V (n=18) and grade VI (n=7). The apoptotic index was high (mean 30%) in advanced lesions compared with controls (<2%) and smooth muscle cells (SMCs) were the predominant cell type undergoing apoptosis. In all TUNEL‐positive apoptotic cells, Bax and Bak were present, while Bcl‐x was absent. Bcl‐2 was absent in a majority of these cells, but occasional TUNEL‐positive cells expressed Bcl‐2. In non‐apoptotic cells, Bcl‐x was present and western blot detected only the long isoform, Bcl‐xL, from the plaques. In conclusion, increased Bax and Bak coupled with lack/paucity of Bcl‐2 and Bcl‐xL are associated with SMC apoptosis in advanced lesions. Bcl‐xL in non‐apoptotic cells appears to contribute to prolonged cell survival. Copyright

Distinct B-cell clonal bands in Helicobacter pylori gastritis with lymphoid hyperplasia

Helicobacter pylori (Hp)‐associated gastritis is a risk factor for gastric mucosa‐associated lymphoid tissue (MALT) lymphoma. Clonal B‐cell populations are present in both reactive and neoplastic MALT tissue, thus limiting their usefulness in the evaluation of gastric lymphoid infiltrates in endoscopic biopsy specimens. The aim of this study was to identify the presence of clonal B‐cell populations in Hp‐gastritis with MALT and to assess their usefulness in distinguishing reactive from malignant infiltrates. Routinely fixed paraffin‐embedded blocks from 20 patients with Hp‐gastritis with lymphoid hyperplasia were analysed for B‐cell clonality by a semi‐nested polymerase chain reaction (PCR) using FRIII/LJH and FRIII/VLJH primers for amplification of the VDJ region of the immunoglobulin heavy chain gene. The histopathological findings were evaluated according to a previously published scoring system. Immunohistochemistry was performed by the labelled streptavidin–biotin technique using the following primary antibodies: CD45, CD45RO, CD3, CD20, and cytokeratin. The histopathological findings were diagnostic of Hp‐chronic active gastritis (grade 2, n=17; grade 3, n=3). Scattered intraepithelial B‐cells were present in all cases and non‐destructive lymphoepithelial lesions in one grade 3 case. Amplifiable DNA was obtained from all samples. Clonal bands were observed in ten (7/17 grade 2 and 3/3 grade 3 lesions) and polyclonal smears in ten cases (all grade 2). The clonal bands were often (n=6) associated with a background polyclonal smear and were not reproducible from deeper sections (n=10) or another paraffin block (n=1), while the clonal bands in control low‐grade MALT lymphomas were not associated with a background smear and were reproducible from deeper sections. None of the patients has developed lymphoma to date (follow‐up 21–44 months). In conclusion, B‐cell clonal bands are common in H. pylori‐gastritis with lymphoid hyperplasia. The irreproducibility of these bands is a useful feature in favouring a reactive process. Copyright

Disrupted RabGAP function of the p85 subunit of phosphatidylinositol 3-kinase results in cell transformation.

Rab proteins regulate vesicle fusion events during the endocytosis, recycling, and degradation of activated receptor tyrosine kinases. The p85α subunit of phosphatidylinositol 3-kinase has GTPase-activating protein activity toward Rab5 and Rab4, an activity severely reduced by a single point mutation (p85-R274A). Expression of p85-R274A resulted in increased platelet-derived growth factor receptor (PDGFR) activation and downstream signaling (Akt and MAPK) and in decreased PDGFR degradation. We now report that the biological consequences of p85-R274A expression cause cellular transformation as determined by the following: aberrant morphological phenotype, loss of contact inhibition, growth in soft agar, and tumor formation in nude mice. Immunohistochemistry shows that the tumors contain activated PDGFR and high levels of activated Akt. Coexpression of a dominant negative Rab5-S34N mutant attenuated these transformed properties. Our results demonstrate that disruption of the RabGAP function of p85α due to a single point mutation (R274A) is sufficient to cause cellular transformation via a phosphatidylinositol 3-kinase-independent mechanism partially reversed by Rab5-S34N expression. This critical new role for p85 in the regulation of Rab function suggests a novel role for p85 in controlling receptor signaling and trafficking through its effects on Rab GTPases.