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Mitochondrial bioenergetics is dynamically coupled with neuronal activities, which are altered by hypoxia-induced respiratory neuroplasticity. Here we report structural features of postsynaptic mitochondria in the pre-Botzinger complex (pre-BotC) of rats treated with chronic intermittent hypoxia (CIH) simulating a severe condition of obstructive sleep apnea. The subcellular changes in dendritic mitochondria and histochemistry of cytochrome c oxidase (CO) activity were examined in pre-BotC neurons localized by immunoreactivity of neurokinin 1 receptors. Assays of mitochondrial electron transport chain (ETC) complex I, IV, V activities, and membrane potential were performed in the ventrolateral medulla containing the pre-BotC region. We found significant decreases in the mean length and area of dendritic mitochondria in the pre-BotC of CIH rats, when compared to the normoxic control and hypoxic group with daily acute intermittent hypoxia (dAIH) that evokes robust synaptic plasticity. Notably, these morphological alterations were mainly observed in the mitochondria in close proximity to the synapses. In addition, the proportion of mitochondria presented with enlarged compartments and filamentous cytoskeletal elements in the CIH group was less than the control and dAIH groups. Intriguingly, these distinct characteristics of structural adaptability were observed in the mitochondria within spatially restricted dendritic spines. Furthermore, the proportion of moderately to darkly CO-reactive mitochondria was reduced in the CIH group, indicating reduced mitochondrial activity. Consistently, mitochondrial ETC enzyme activities and membrane potential were lowered in the CIH group. These findings suggest that hypoxia-induced respiratory plasticity was characterized by spatially confined mitochondrial alterations within postsynaptic spines in the pre-BotC neurons. In contrast to the robust plasticity evoked by dAIH preconditioning, a severe CIH challenge may weaken the local mitochondrial bioenergetics that the fuel postsynaptic activities of the respiratory motor drive.

Extraskeletal osteosarcoma is a rare, highly malignant, osteoid formation mesenchymal neoplasm in the absence of bone involvement, associated with exceptionally poor prognosis. It frequently arises in the soft tissues of the extremities or in the retroperitoneum, but rarely in visceral organ. We describe a primary osteosarcoma of the liver in a 70-year-old man who presented with an episode of fever, accompanied by abdominal discomfort, after an accident abdominal strike. Ultrasonography and computed tomography revealed a large heterogeneous mass with areas of dense calcification involving most of the right lobe of liver. Radiography did not show evidence of primary tumor or primary bone lesion at any other site. Histologically, the tumor showed an essentially similar appearance as osteosarcoma originating in the skeleton, comprised of polygonal or spindle shaped cells, along with abundant eosinophilic lace-like osteoids, or irregularly arranged bone trabeculae. Immunohistochemistry showed that the tumor cells were positive for vimentin, CD10, and focally for SMA and CD56, but negative for other lineage-specific markers. Thus, the findings favored a primary hepatic osteosarcoma. This patient received palliative chemotherapy to ease the signs of his sickness due to the large size of the tumor and he died 4 moths later.

Follicular dendritic cell (FDC) sarcoma, especially of extranodal origin, is an extremely rare malignancy of FDC origin, with only 1 case previously documented in the thyroid. The authors report the case of a 58-year-old female who presented with a painless mass in her neck. The neoplastic cells expressed monocyte/macrophage-specific marker CD68 (KP-1) and lysozymes and the dendritic cell-associated antigens CD35 and Fascin but was negative for CD1a, CD21, and CD23, most consistent with a diagnosis of FDC sarcoma. BIOMED-2 multiplex polymerase chain reaction analysis showed B-cell clonality in both tumor and its adjacent coexisting Hashimoto’s thyroiditis. To the authors’ knowledge, this is the first report of a rare entity of FDC sarcoma primarily involving the thyroid gland coexisting with Hashimoto’s thyroiditis.

Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein. The levels of HTNV NP-specific antibody and cellular immune response were measured by use of ELISA, fluorescence activated cell sorter (FACS) analysis, cytotoxicity assay, and IFN-gamma ELISPOT assay. We found that HTNV S-HSP70 DNA vaccination significantly increased the levels of HTNV NP-specific antibody, IgG2a/IgG1 ratio, IFN-gamma producing CD8+ T-cell precursor frequencies, and cytotoxic T lymphocyte (CTL) response when compared with immunization with HTNV S DNA alone or HTNV S DNA physically mixed with HSP70 DNA. By contrast, HSP70 DNA or vector DNA immunization could not induce appreciable levels of specific antibodies and CTL response. Thus, we demonstrate for the first time that HSP70-based HTNV S DNA can induce both humoral and cellular immune response specific for HTNV NP and is a promising candidate DNA vaccine for HTNV infection.

Pleuropulmonary blastoma (PPB) is a rare malignant dysontogenetic neoplasm primarily affecting younger children, even in newborns with an unfavorable outcome. PPB is histologically composed of a primitive, variably mixed blastematous and sarcomatous components, and exclusively subclassified as type I (purely cystic), type II (both cystic and solid elements) and type III (completely solid) by increasing histological evidence of malignancy. At present, well-documented cases or cases of truly precise presentation of either pathological or immunohistochemical findings in PPB are rare. The authors report one case of PPB in a 44-month-old child presenting as a solid and cystic mass with special emphasis on its radiological, histopathological and immunohistochemical aspects. The histological diagnosis was PPB, which would belong to the type II category.

Kaposiform hemangioendothelioma (KHE) is a rare locally aggressive vascular neoplasm characterized by infiltrating nodules and sheets of spindle cells, and unmistakable resemblance to Kaposi's sarcoma. KHE occurs mainly in newborns and infants and presents most commonly in the skin, deep soft tissue, and bone. We report a case of KHE in a 36-year-old female who presented with a spleen mass and underwent splenectomy. Macroscopic examination revealed a large, dark-red, firm mass in the spleen. Histologically, the tumor consisted of irregular, infiltrating nodules of densely packed spindle-shaped tumor cells closely associated with small slit-like and sieve-like blood vessels, which were separated with hyalinized hypocellular fibrous stroma. Immunohistochemically, both spindle and epithelioid cells were positive for CD34, CD31, and vimentin, but negative for EMA, cytokeratin, CD21, CD35, CD1a, and S-100 protein. The well-formed capillaries and mature vessels but not spindle tumor cell showed reactivity for factor VIII- related antigen. Alpha-Smooth muscle actin was detected in pericytes surrounding small round or slit-like capillaries. The final histologic diagnosis was KHE. Follow-up 6 month after operation revealed no sign of recurrence or metastasis.To the best of our knowledge, this is the first report of KHE arising in the spleen.

Histiocytic sarcoma (HS) is an extremely rare true histiocytic malignancy. We report a case of HS arising from thyroid gland in a 69 year-old man. Following subtotal thyroidectomy, a histopathologic, immunohistologic, and genotypic examination revealed HS. This tumor was composed of large spindle or round epithelioid cells with abundant eosinophilic cytoplasm. The neoplastic cells were positive for macrophage-associated antigen CD68, CD163, and lysozymes, as well as CD45, HLA- DR, DP, DQ, and S100, most consistent with a diagnosis of HS. The BIOMED-2 multiplex PCR analysis showed polyclonal B- and T-cell populations. To our knowledge, this is the first report of a rare entity HS involving thyroid gland using a comprehensive immunophenotyping panel including CD163 as well as molecular studies to establish the true histiocytic nature of these lesions.

To synthesize p53 mutants 249M, 273H with site-directed mutagenesis and construct the relative GFP expression vectors, transfect HepG2 cells and then detect the co-localization of p53 and hsp70.Two sets of primers were designed according to the gene sequence of p53, and mismatches were introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments containing the mutation site from the second PCR were subcloned into the pEGFP-C3 vectors. The localization of p53 and hsp70 was observed in transfected HepG2 cells using confocal microscopy.The sequencing analysis showed that the mutated sites were correct. The expression vectors were constructed successfully. Endogenous hsp70 co-localized in cytoplasm with 273H p53, wt p53 and 249M p53 localized in nucleus.We have found p53 and hsp70 positive products were colocalized in nuclei of human HCC, while the endogenous hsp70 were colocalized in cytoplasm with 273H p53 in HepG2 cells. This contradictory phenomenon suggests that perhaps there are unclear mechanisms existed between the relationship of hsp70 and p53.

Human nuclear respiratory factor 2 alpha subunit (NRF-2α) is fundamentally important to cell function and the development. We aimed to establish the monoclonal antibody (MAb) against the human NRF-2α protein and to investigate its distribution in human hepatocellular carcinoma (HCC) and tumor-adjacent tissues. The 6× His-NRF-2α fusion protein was successfully induced and purified. One monoclonal antibody (MAb) against human NRF-2α, 1-D10-E1-B11-G3 (IgG1), effective in detecting the recombinant and the cellular protein, was characterized. Using immunohistochemical analysis, the expression of NRF-2α was investigated in 38 cases of HCC specimens and 14 cases of tumor-adjacent specimens. Staining was found positive in 9 cases of HCC tissues (23.7%) and 8 cases of normal hepatic tissues (57.1%). The higher-grade frequency of expression of NRF-2α in tumor-adjacent tissues was significantly higher (P < 0.01) than that in tumor tissues, suggesting that NRF-2α may play important roles in carcinogenesis of HCC.

To explore the expression and significance of heat shock protein 70 (Hsp70), glucose regulated protein 94 (Grp94) and immunoglobulin G (IgG) in human lung carcinoma.The expression of Hsp70, Grp94 and IgG in 40 human lung carcinomas was studied using immunohistochemical technique and image analysis. The localization among Hsp70, Grp94 and IgG was analyzed by double labeling immunofluorescent staining and laser scanning confocal microscopy.Hsp70, Grp94 and IgG in Human lung carcinomas showed high expression. The positive rate of Hsp70, Grp94 and IgG was 65% (26/40), 45% (18/40), and 82.5% (33/40), respectively. The average value of optical density was 5.10 +/- 0.32, 3.52 +/- 0.35, and 8.12 +/- 0.31, respectively. Hsp70 was localized in nucleus and cellular cytoplasm while Grp94 and IgG were mainly localized in cellular cytoplasm. Ten cases showed Hsp70 was co-localized with IgG and Eighteen cases showed Grp94 was co-localized with IgG.High expression of Hsp70, Grp94 and IgG in Human lung carcinomas suggested that Hsp70, Grp94 and IgG might play an important role in the development of human lung carcinoma. IgG is co-localized with Hsp70 or Grp94. The study will lay a theorical basis for further research on anti-tumor immunotherapy.

Summary Heat shock proteins (HSPs) are known to act as an effective molecular adjuvant to enhance the induction of antigen peptide-specific cellular immunity, when coupled with the antigen or peptide. Hantaan virus (HTNV) nucleocapsid protein (NP) is relatively conserved among hantaviruses and highly immunogenic in both animals and humans. To analyze the influence of HSP70 on NP vaccine potency, and evaluate the possibility of developing a novel effective vaccine against hantaviruses, we constructed prokaryotic expression plasmids, and expressed three recombinant proteins, namely, HTNV NP, HSP70 and HSP70 − NP fusion protein. As an alternative to fusion protein, we also generated HSP70 and HTNV NP complexes (HSP70 + NP) in vitro. C57BL/6 mice were immunized with those recombinant proteins, the humoral and cellular responses elicited against NP were measured by ELISA, fluorescence flow cytometry, cytotoxicity assays, and IFN-γ ELISPOT assay. We found that immunization of mice with HSP70 − NP fusion protein, or HSP70 + NP complexes elicited significantly higher NP-specific antibody titers, frequencies of IFN-γ-producing cells and cytotoxic T lymphocyte (CTL) activities in vivo than conventional HTNV NP vaccination. Antibody isotype analysis showed that the antibody response was characterized by a higher HTNV NP-specific titer of IgG2a than IgG1 antibodies, resulting in a significant higher IgG2a/IgG1 ratio. By comparison, HSP70 − NP fusion protein is significantly superior to HSP70 + NP complexes in enhancement of NP antigenicity. These results indicated that HSP70, when fused to or complexed with HTNV NP, greatly enhance NP vaccine potency by preferential induction of a predominant Th1 immune response in a NP-specific, HSP70-dependent manner.

To study stress response in human umbilical vein endothelial cells (HUVEC) infected with Hantavirus (HTV).The expression of HSP70 was detected by immunocytochemical staining, in situ nucleic acid hybridization and RT-PCR.After being infected with HTV, the expression of HSP70 in HUVEC increased. RT-PCR showed that the level of HSP70 mRNA increased rapidly and kept at a high level in comparison with the normal control (P0.05).The over-expression of HSP70 in HUVECs can be induced by Hantavirus infection, which may play a role in the inhibition of virus reproduction and the protection of cells from virus infection.

The macaque visual cortex is exquisitely organized into columns, modules, and streams, much of which can be correlated with its metabolic organization revealed by cytochrome oxidase (CO). Plasticity in the adult primate visual system has also been documented by changes in CO activity. Yet, the molecular mechanism of regulating this enzyme remains not well understood. Being one of only four bigenomic enzymes in mammalian cells, the transcriptional regulation of this enzyme necessitates a potential bigenomic coordinator. Nuclear respiratory factor 2 (NRF-2) or GA-binding protein is a transcription factor that may serve such a critical role. The goal of the present study was to determine if the two major subunits of NRF-2, 2α and 2β, had distinct subcellular distribution in neurons of the rat and monkey visual cortex, if major metabolic neuronal types in the macaque exhibited different levels of the two subunits, and if they would respond differently to monocular impulse blockade. Quantitative immuno-electron microscopy was used. In both rats and monkeys, nuclear labeling of α and β subunits was mainly over euchromatin rather than heterochromatin, consistent with their active participation in transcriptional activity. Cytoplasmic labeling was over free ribosomes, the Golgi apparatus, and occasionally the nuclear envelope, signifying sites of synthesis and possible posttranslational modifications. The density of both subunits was much higher in the nucleus than in the cytoplasm for all neurons examined, again indicating that their major sites of cellular action is in the nucleus. In both layer IVC and supragranular puffs of the macaque visual cortex, the expression of both NRF-2α and β was higher in medium-sized, non-pyramidal (type C and C-like) cells previously shown to have higher CO activity than small, type A and A-like cells with low CO activity. Pyramidal, type B cells in puffs had intermediate levels of CO as well as NRF-2α and β labeling. Monocular impulse blockade induced a greater reduction of NRF-2 labeling in type C/C-like than type A/A-like cells. These results substantiate and extend our previous findings that NRF-2 is constitutively active in adult primate and rat visual cortical neurons, that it is expressed more strongly in metabolically more active neurons, and that its level is directly regulated by neuronal activity, the blockade of which imposes a greater down-regulation of this transcription factor in metabolically more active than less active neurons.

To investigate the interaction between p53 and heat shock protein70 (HSP70) in human hepatocellular carcinoma tissues.HSP70 and p53-double positive samples were screened by imunohistochemical staining from twelve human hepatocellular carcinoma tissues, and the tissue homogenate was prepared. Coimmunoprecipitation was performed by using either anti-HSP70 mAb or anti-p53 mAb. The immunoprecipitate was analysed by SDS-PAGE and Western blot.Immunohistochemical staining detection showed that three samples out of twelve cases were double positive for both HSP70 and p53. p53 protein existed in the immunoprecipitate of anti-HSP70 mAb, while there was HSP70 protein in the immunoprecipitate of anti-p53 mAb.HSP70 and p53 exist as a complex in human hepatocellular carcinoma tissues, which will provide a new route for studying the pathogenesis and immunotherapy of human hepatocellular carcinoma.

To obtain high therapeutic effect and low toxicity single-chain immunotoxin against hepatocellular carcinoma (HCC).Human mutant tumor necrosis factor-alpha (mTNFalpha) was linked with the 3' end of humanized single-chain Fv against HCC (hscFv25) in pGEX4T-1 vector. The anti-HCC immunotoxin was expressed in Escherichia coli and identified by western blot. The primary tumor regression trial in nude mice bearing HCC was evaluated the targeting therapeutic value of hscFv25-mTNFalpha. The tumor tissues were stained by immunohistochemical with TNFalpha antibody.The expression of single-chain immunotoxin hscFv25-mTNFalpha was 12% of total bacteria proteins. The tumor regression trials of hscFv25-mTNFalpha showed 5/5 effective. It had 2/5 completely remission and 3/5 partly remission. The therapeutic result of hscFv25-mTNFalpha was better than that of mTNFalpha (F=8.70, 0.05). The HCC tissue treated by hscFv25-mTNFalpha expressed TNFalpha positive reaction. The positive granule mainly existed in HCC cytoplasm.The single-chain immunotoxin hscFv25-mTNFalpha has high therapeutic effect and low toxicity. It has potentialities for clinical application.

Nuclear respiratory factor (NRF)-2 or GA-binding protein is a potential transcriptional, bigenomic coordinator of mitochondrial and nuclear-encoded subunits of cytochrome oxidase genes. It is composed of an alpha subunit that binds DNA and a beta subunit that has the transactivating domain. Previously, we found that the level of NRF-2 paralleled that of cytochrome oxidase under normal and functionally altered states. The goal of our present study was to increase the resolution to the ultrastructural level and to quantify changes before and after depolarizing stimulation. We used a pre-embedding immunogold-silver method for the two subunits of NRF-2 in cultured rat visual cortical neurons. NRF-2alpha and beta were normally located in both the nucleus and the cytoplasm. In the nucleus, both subunits were associated primarily with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, they were associated mainly with free ribosomes and occasionally with the Golgi apparatus and the outer membrane of the nuclear envelope. Labelling was not found in the mitochondria, confirming the specificity of the antibodies. Neuronal depolarization by KCl for 5 h induced a six- to seven-fold increase in the nuclear-to-cytoplasmic ratio of both subunits (P < 0.001) without increases in total labelling densities. These results strongly indicate that both NRF-2alpha and NRF-2beta respond to increased neuronal activity by translocating from the cytoplasm to the nucleus, where they engage in transcriptional activation of target genes. Our results also indicate that the cytoplasmic to nuclear movement of transcription factors is a dynamic process induced by neuronal activity.

To obtain human recombinant Fv-immunotoxin hscFv(25)-mTNFalpha (mutant human TNFalpha fused to human scFv(25)) against hepatocellular carcinoma (HCC).Two relevant sites of enzymatic digestion were added to mTNFalpha by PCR. MTNFalpha was linked to the 3' end of hscFv(25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was expressed in Escherichia coli and purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins. MTNFalpha protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFalpha antibody.The expression ratio of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was 12% of bacterial protein. The result of tumor restraining trials of hscFv(25)-mTNFalpha showed 2/5 complete remission and 3/5 partial remission. mTNFalpha restraining trials showed 5/5 partial remission. The therapeutic result of hscFv(25)-mTNFalpha was better than that of mTNFalpha (F=8.70, P0.05). The hscFv(25)-mTNFalpha remedial tumor tissues were positive for TNFalpha by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell.Recombinant Fv-immunotoxin hscFv(25)-mTNFalpha has better therapeutic effect than mTNFalpha. It can inhibit the cellular growth of HCC and has some potential of clinical application.