(18)F-FDG PET/CT in Patients with Nodular Pulmonary Amyloidosis: Case Report and Literature Review.

A 62-year-old woman was found to have multiple bilateral pulmonary nodules showing different (18)F-fluorodeoxyglucose (FDG) uptakes on positron-emission tomography/computed tomography (PET/CT). Only the largest nodule in the left lower lobe showed an increased (18)F-FDG uptake on PET/CT. Three nodules were surgically resected from different lobes of the left lung. Two lobes were benign and showed amyloid deposition. The largest nodule in the left lower lobe showed adenocarcinoma and a heavy amyloid deposition. Pulmonary amyloidosis should be added to the differential diagnosis for cases with multiple pulmonary nodules that show different (18)F-FDG uptakes on PET/CT. To the best of our knowledge, this is the second reported case of a lung nodule consisting of adenocarcinoma and amyloid deposition.

Up-regulation of cyclin D1 expression in asthma serum-sensitized human airway smooth muscle promotes proliferation via protein kinase C alpha.

Abnormal hypertrophy and hyperplasia of airway smooth muscle cells play an important role in airway remodeling in chronic asthma. The authors' previous studies have indicated that protein kinase C alpha (PKC alpha) is involved in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). However, the underlying mechanisms remain unknown. Here, the authors examined the possible role of the alpha isoform of PKC in the control of cyclin D1 expression, using HASMCs passively sensitized on human atopic asthmatic serum as a model system. Cultured HASMCs were passively sensitized with serum from atopic asthmatic patients. Cell proliferation was measured by [(3)H]thymidine incorporation and an MTT assay. Cell cycle status was analyzed by flow cytometry. The mRNA and protein expression profiles of cyclin D1 were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, the authors assessed the role of cyclin D1 in PKC alpha-induced HASMC proliferation by transfection with a recombinant cyclin D1 antisense construct. The activation of PKC alpha with phorbol myristate acetate (PMA), a PKC activator, up-regulated cyclin D1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly decreased by specific inhibition of PKC alpha with Go6976. In addition, the authors showed that transfection with antisense cyclin D1 abolished PMA-induced G1/S progression and HASMC proliferation. These results demonstrate that PKC alpha promotes the proliferation of HASMCs sensitized with atopic asthmatic serum via up-regulation of cyclin D1 expression.

Role of protein kinase C alpha and cyclin D1 in the proliferation of airway smooth muscle in asthmatic rats.

BACKGROUND: Airway smooth muscle (ASM) is suspected to be a determining factor in the structural change of asthma. However, the role of protein kinase C alpha (PKCalpha) and cyclin D1 involved in the dysfunction of ASM leading to asthmatic symptoms is not clear. In this study, the central role of PKCalpha and cyclin D1 in ASM proliferation in asthmatic rats was explored. METHODS: Thirty-six pathogen-free male Brown Norway (BN) rats were randomly divided into 2 groups: control groups (group N1, N2 and N3) and asthmatic groups (group A1, A2, and A3). Groups A1, A2 and A3 were challenged with ovalbumin (OA) for 2 weeks, 4 weeks and 8 weeks respectively. Control animals were exposed to an aerosolized sterile phosphate buffered saline (PBS). The ASM mass and nucleus numbers were studied to estimate the degree of airway remodeling by the hematoxylin-eosin staining method. PKCalpha and cyclin D1 expression in the ASM cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The relation between PKCalpha and cyclin D1 was assessed by linear regression analysis. PKC agonist phorbol 12-myristate 13-acetate (PMA), PKC inhibitor Ro31-8220 and an antisense oligonucleotide against cyclin D1 (ASOND) were used to treat ASM cells (ASMCs) obtained from the 2 weeks asthmatic rats. The cyclin D1 protein expression level was detected by Western blotting. RESULTS: Compared with the control group, the PKCalpha and cyclin D1 mRNA levels were increased in the asthmatic group. Similar to RT-PCR results, immunohistochemistry analysis for PKCalpha and cyclin D1 expression revealed an increased production in ASMCs after allergen treatment for 2, 4 and 8 weeks compared with the respective control groups. No difference in expression of PKCalpha and cyclin D1 in ASM were found in the 2, 4 or 8 weeks asthmatic rats. There were significant positive correlations between PKCalpha and cyclin D1 expression, both transcriptionally (r = 0.944, P < 0.01) and translationally (r = 0.826, P < 0.01), in ASM. The content of cyclin D1 in asthmatic ASMCs increased after being stimulated by PMA, and decreased when induced by Ro31-8220. ASOND targeting for cyclin D1 lowered the expression of cyclin D1 induced by PMA. CONCLUSIONS: Increased expression of PKCalpha and cyclin D1 in ASM along with smooth muscle structure changes might implicate PKCalpha and cyclin D1 participation in the proliferation of ASM and contribute to the pathogenesis of asthma after repeated allergen exposure in rats. The results suggested that cyclin D1 might be downstream of PKC signal transduction pathway.

[Inhibition of NF-kappaB through IkappaBalpha transfection affects invasion of human lung cancer cell line A549].

BACKGROUND & OBJECTIVE: Recent studies have shown that activation of nuclear factor-kappaB(NF-kappaB) can regulate the invasion and metastasis of cancer cells. The present study was to investigate inhibition of NF-kappaB activity on invasion of human lung cancer cell line A549 and the possible mechanism. METHODS: The recombinant plasmid pcDNA3.1(+)/IkappaBalpha, expressing the alpha isoform (IkappaBalpha) of the NF-kappaB inhibitor, was constructed. A549 cells were cultured in vitro and divided into non-transfection group, pcDNA3.1(+) transfected group and pcDNA3.1(+)/IkappaBalpha transfected group. The expression of IkappaBalpha was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The activity of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA). Invasion of A549 cells was assessed by transwell chamber assay. The expression of MMP-2 and MMP-9 was detected by RT-PCR and gelatin zymography. RESULTS: Plasmid pcDNA3.1(+)/IkappaBalpha was successfully constructed and expressed in A549 cells. The activity of NF-kappaB, the number of invasive cells, the activity of MMP-2 and MMP-9 of A549 cells in pcDNA3.1(+)/IkappaBalpha transfected group were significantly lower than those in non-transfection group and pcDNA3.1(+) transfected group (all P<0.05). CONCLUSION: Transfection of IkappaBalpha can inhibit NF-kappaB activity, thus inhibit cell invasion of A549, which may be through the down-regulation of MMP-2 and MMP-9 expressions.

[Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats].

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

Role of Shenfu Injection in rats with systemic inflammatory response syndrome.

OBJECTIVE: To investigate the role of Shenfu Injection (SFI) in rats with systemic inflammatory response syndrome (SIRS). METHODS: The SIRS rat model was induced by the intravenous injection of lipopolysaccharide (LPS). Forty-five male Wistar rats were randomly divided into 3 groups, the sham operative control group (control group, n=5), the SIRS model group (model group, n=20) and the SFI treatment group (SFI group, n=20). LPS was injected through the external jugular vein (12 mg/kg, 6 mg/mL) to all rats except for those in the control group, and SFI (10 mL/kg) was given to those in the SF group only once through intraperitoneal injection, while the normal saline (10 mL/kg) was given to those in the model group. For those in the control group, normal saline was given through the external jugular vein (2 mL/kg) and intraperitoneal injection (10 mL/kg). Then, rats in the model group and SFI group were divided into 4 subgroups according to the time points, i.e., 1 h, 2 h, 4 h and 6 h subgroups, 5 rats in each group. The activity of nuclear factor of kappa B (NF-kappa B) of in blood mononuclear cells and the plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6-(IL-6) were determined using enzyme-linked immunoabsordent assay (ELISA) at 1 h, 2 h, 4 h and 6 h after modeling. Histopathologic changes of the lung and liver were observed under a light microscope. RESULTS: Compared with the control group, the activity of NF-kappa B in mononuclear cells and the plasma level of TNF-alpha were obviously increased at each time points (all P<0.01), reaching the peaks at 2 h after modeling. The plasma level of IL-6 increased gradually as time went by in the model group (P<0.01). Pathological examination showed pulmonary alveoli hemorrhage, edema and inflammatory cell infiltration in the lung tissue, and angiotelectasis, congestion, and local necrosis in the liver tissue in the model group. Compared with the model group, the activity of NF-kappa B and the levels of TNF-alpha and IL-6 in plasma decreased significantly in the SFI group (P<0.01), and the pathological injury in the lungs and liver was significantly alleviated. CONCLUSION: SFI plays a protective role by inhibiting the activity of NF-kappaB, and reducing the expressions of TNF-alpha and IL-6 in SIRS rats.

PKC promotes proliferation of airway smooth muscle cells by regulating cyclinD1 expression in asthmatic rats.

AIM: To determine whether protein kinase C (PKC) has any effect on the expression of cyclinD1, a key regulator of growth control and G1/S transition, and to investigate the underlying molecular mechanisms of PKC involving the remodeling of the asthmatic airway smooth muscle (ASM). METHODS: The treatment of synchronized ASM cells from asthmatic rats with PKC-specific agonist phorbol 12-myristate 13-acetate (PMA) and antagonist 2-{1-[3-(amidinothio) propyl]-1Hindol-3-yl}-3-(1-methylindol-3-yl) maleimide methanesulfonate salt (Ro31-8220) was followed by the proliferation assay. PKCalpha and cyclinD1 expressions in ASM cells (ASMC) were detected by RT-PCR and Western blotting. The relation between PKCalpha and cyclinD1 was assessed by linear regression analysis. The effect of the construct recombinant plasmid pcDNA3.1-antisense cyclinD1 (pcDNA3.1-ascyclinD1) on the proliferation of ASMC was found to be induced by PMA. RESULTS: The data showed phorbol ester-dependent PKCalpha promoted the proliferation of ASMC. The closely-positive correlation existed between the expression of PKCalpha and cyclinD1 at the transcriptional (r=0.821, P<0.01) and translational (r=0.940, P<0.01) levels. pcDNA3.1-ascyclinD1 could inhibit the proliferation of ASMC. pcDNA3.1-ascyclinD1 almost completely attenuated the PMA-induced proliferation effect as Ro31-8220+pcDNA3.1. CONCLUSION: The proliferation of ASMC by PKC might by regulated by the cyclinD1 expression in asthmatic rats.

[PKCalpha-ERK1/2 cascade is involved in up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells sensitized by sera from atopic asthmatics].

OBJECTIVE: To explore the role of PKCalpha-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells (HASMCs) sensitized by sera from atopic asthmatics. METHODS: HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups: the control group, PMA treated group, PMA and PKCalpha mismatched Oligodeoxynucleotides (PKCalpha-mmODN) treated group, PMA and PKCalpha antisense Oligodeoxynucleotides (PKCalpha-asODN) treated group, PMA and U0126 (MAP Kinase Kinase inhibitor)treated group. The expression of p-PKCalpha, ERK1/2, p-ERK1/2, cyclinD1 and P21(cip1) protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTT colorimetric assay. RESULTS: Compared with the control group, the expression of p-PKCalpha and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21(cip1) protein increased correspondingly (the A value % control was 2.10 +/- 0.29, 1.67 +/- 0.19, 2.20 +/- 0.27, 1.99 +/- 0.22 and 3.11 +/- 0.29 respectively; q value was 9.87, 7.06, 10.57, 11.10 and 20.33 respectively; all P < 0.05) in PMA treated group, and cells proliferation [the percentage of cells in S phase was (30.3 +/- 2.4)%, A(490) value was 0.80 +/- 0.06] enhanced significantly compared with those [the percentage of cells in S phase was (13.9 +/- 2.6)%, A(490) value was 0.41 +/- 0.04] of the control group (q = 6.07, 12.63; all P < 0.05). In PMA and PKCalpha-asODN treated group, the level of p-PKCalpha decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21(cip1) decreased correspondingly (the A value % control was 1.23 +/- 0.19, 1.34 +/- 0.18, 1.52 +/- 0.20, 1.45 +/- 0.18 and 1.49 +/- 0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (21.2 +/- 2.8)%, A(490) value was 0.51 +/- 0.04; q = 6.07, 12.63; all P < 0.05], as compared with those of the PMA treated group. In PMA and U0126 treated group, the level of p-PKCalpha had no significant change (A value was1.99 +/- 0.18, q = 0.94, P > 0.05), but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21(cip1) reduced (the A value % control was 0.95 +/- 0.21, 1.15 +/- 0.19, 1.37 +/- 0.15 and 1.96 +/- 0.21 respectively; q value was 7.79, 9.16, 6.92 and 11.16 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (22.0 +/- 3.2)%, A(490) value was 0.49 +/- 0.03; q = 5.51, 13.45; all P < 0.05], as compared with those of the PMA treated group. CONCLUSION: ERK1/2 is one of the downstream regulators of PKCalpha, and PKCalpha-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21(cip1) and proliferation in HASMC sensitized by sera from atopic asthmatics.

[Nuclear factor-kappaB activity and its correlation with cell proliferation in non-small cell lung cancer tissues].

OBJECTIVE: To investigate the nuclear factor (NF)-kappaB activity in human non-small cell lung cancer (NSCLC) tissues and to explore the correlation between NF-kappaB activity and cell proliferation, between NF-kappaB activity and cell spontaneous apoptosis. METHODS: Thirty samples of non-small cell lung cancer tissues and 15 normal lung tissues were collected from May to October in 2006. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). CyclinD1 level was examined by RT-PCR and Western blot. Proliferation cell nuclear antigen (PCNA) protein was examined by immunohistochemical analysis. Spontaneous cell apoptosis was determined by the TUNEL method. RESULTS: There was significant difference (F=78.96, P<0.01) in NF-kappaB activity among normal lung tissue group (24,826+/-3724), squamous-cell carcinoma tissue group (28,028+/-4204), and adenocarcinoma tissue group (35,425+/-5317). The NF-kappaB activity in the squamous-cell carcinoma group and the adenocarcinom tissue group was higher than that in the normal lung tissue group (all P<0.01); and the NF-kappaB activity is in the adenocarcinoma tissue group was higher compared with that in the squamous-cell carcinoma group (P<0.05). There was significant difference (F=62.43, P<0.01) in the cyclinD1 mRNA level among normal lung tissue group (2.04+/-0.24), the squamous-cell carcinoma group (2.91+/-0.37), and the adenocarcinoma group (4.13+/-0.36). There was significant difference (F=89.24, P<0.01) in cyclinD1 protein level among normal lung tissue group (0.31+/-0.06), the squamous-cell carcinoma group (0.43+/-0.07), and the adenocarcinoma group (0.58+/-0.08). There was significant difference (F=45.61, P<0.01) in PCNA protein level among the normal lung tissue group (0.32+/-0.09), the squamous-cell carcinoma group (0.42+/-0.10), and the adenocarcinima group (0.54+/-0.16). There was no significant difference (F=1.86, P>0.05) in apoptosis index among the normal lung tissue group (2.58%+/-0.39%), the squamous-cell carcinoma group (2.27%+/-0.34%), and the adenocarcinoma group (2.92%+/-0.59%). The NF-kappaB activity was positively correlated with cyclin D1 mRNA level, cyclin D1 protein level, and PCNA protein level in the squamous-cell carcinoma group (r=0.51, P<0.05, r=0.54, P<0.05, r=0.60, P<0.05), respectively; the NF-kappaB activity was also positively correlated with cyclinD1mRNA, cyclinD1protein level, and PCNA protein level in the adenocarcinoma group (r=0.60, P<0.05; r=0.64, P<0.05; r=0.68, P<0.05), respectively. The NF-kappaB activity in the squamous-cell group and the adenocarcinoma group was not related to cell apoptosis index. CONCLUSION: NF-kappaB activity increased in NSCLC tissues. Abnormal NF-kappaB activation may be associated with cell proliferation, but do not affect spontaneous cell apoptosis in NSCLC tissues.