p-CREB-1 at Ser 133 is a potential marker for breast cancer.

OBJECTIVE: Dysregulation of numerous oncogenes and their downstream signaling pathways, among others in the signaling transduction molecule p-CREB-1 (p-cAMP responsive element binding protein-1), is an essential feature of different types of cancer. To investigate whether p-CREB-1 is also pivotal in tumorigenesis and metastogenesis of breast cancer, we conducted a prospective study with long-term follow-up on 96 patients with breast cancer. PATIENTS AND METHODS: Pathway array and tissue microarray (TMA) were used to detect the differential expression of CREB (cAMP-responsive element binding protein) and p-CREB-1 in breast cancer cells, breast cancer stem cells (BCSCs), human breast cancer tissues (BCTs), and adjacent normal tissues (ANTs). The associations between p-CREB-1 expression, clinicopathological variables, and survival rates of the patients were analyzed and calculated. RESULTS: Our results revealed that p-CREB-1 and CREB expression in cancerous cell lines and tissues were significantly upregulated compared with non-cancerous cell lines and tissues. Most statistically significant overexpression was detected in BCSCs (p<0.01). In TMA and immunohistochemical analyses, BCTs exhibited significantly higher expression of p-CREB-1 and CREB than ANTs (p<0.001). Clinicopathological variable and survival analysis revealed a correlation between high expression (++/+++) of p-CREB-1 and the presence of axillary lymph node metastasis (p<0.05) and poorer disease-free and overall survival. CONCLUSIONS: p-CREB-1 is a potential predictive and prognostic biomarker and a promising therapeutic target in breast cancer.

Atlas-Based Classification of Hyperintense Regions from MR Diffusion-Weighted Images of the Brain: Preliminary Results.

The study of subjects with acquired brain damage in a specific location is important in exploring human brain function. Description of lesion locations within and across subjects is a crucial methodological component that usually involves the distinction of normal from damaged tissue (lesion segmentation) in relation to lesion locations in terms of a standard anatomical reference space (lesion mapping). Our study provides an atlas-based, computer-aided methodology for classification of hyperintense regions on diffusion-weighted images of the brain, representing either ischemic lesions or susceptibility artifacts. We applied a leave-one-out method of cross-validation that computed probabilistic atlases of true lesions and artifacts, based on training data. Our approach accurately classifies lesions and artifacts, but leaves a significant number of regions unclassified, due to the relatively small number of training samples. An initial segmentation step based on a larger sample of data sets is required to automate discrimination of lesions and artifacts.

An abnormal nonhyperdiploid karyotype is a significant adverse prognostic factor for multiple myeloma in the bortezomib era.

Multiple myeloma is clinically heterogeneous and risk stratification is vital for prognostication and informing treatment decisions. As bortezomib is able to overcome several high-risk features of myeloma, the validity of conventional risk-stratification and prognostication systems needs to be reevaluated. We study the survival data of 261 previously untreated myeloma patients managed at our institution, where bortezomib became available from 2004 for the treatment of relapse disease. Patient and disease characteristics, and survival data were evaluated overall, and with respect to bortezomib exposure. Overall, the international staging system (ISS), metaphase karyotyping and interphase fluorescence in situ hybridization (FISH) were discerning of survival outcomes, where the median for the entire cohort was 5.2 years. However, when stratified by bortezomib exposure, only metaphase karyotyping was still discriminating of long-term prognosis. The presence of an abnormal nonhyperdiploid karyotype overrides all other clinical and laboratory parameters in predicting for a worse outcome on multivariate analysis (median survival 2.6 years, P = 0.001), suggesting that bortezomib used at relapse is better able to overcome adverse risk related to high tumor burden (as measured by the ISS) than adverse cytogenetics on conventional karyotyping. Metaphase karyotyping provides additional prognostic information on tumor kinetics where the presence of a normal diploid karyotype in the absence of any high-risk FISH markers correlated with superior survival and could act as a surrogate for lower plasma cell proliferation.

Attainment of at least a very good partial response after induction treatment is an important surrogate of longer survival for multiple myeloma.

The importance of achieving a very good partial response or better (≥VGPR) after induction treatment of myeloma has traditionally only been discussed in the context of high-dose therapy with auto-SCT (HDT/auto-SCT). Of late, the advent of novel agents for induction treatment has resulted in improved CR and VGPR rates, which are comparable with those observed with HDT/auto-SCT. We show that in an unselected group of 179 myeloma patients with diverse baseline characteristics, and treated with different modern induction regimens within a single institution, the attainment of ≥VGPR with or without HDT/auto-ASCT represents a major surrogate marker of better clinical outcomes. On the basis of a 1-year landmark survival analysis, patients achieving ≥VGPR enjoy a significantly longer PFS, which translated to a longer OS. Superseding the adverse effects of advanced age, high International Staging System (ISS) stage, adverse cytogenetics and independent of the transplant status, the attainment of ≥VGPR emerged as the single most significant predictor of long-term survival on multivariate analysis.

Postmenopausal hormone therapy and subclinical cerebrovascular disease: the WHIMS-MRI Study.

OBJECTIVE: The Women's Health Initiative Memory Study (WHIMS) hormone therapy (HT) trials reported that conjugated equine estrogen (CEE) with or without medroxyprogesterone acetate (MPA) increases risk for all-cause dementia and global cognitive decline. WHIMS MRI measured subclinical cerebrovascular disease as a possible mechanism to explain cognitive decline reported in WHIMS. METHODS: We contacted 2,345 women at 14 WHIMS sites; scans were completed on 1,424 (61%) and 1,403 were accepted for analysis. The primary outcome measure was total ischemic lesion volume on brain MRI. Mean duration of on-trial HT or placebo was 4 (CEE+MPA) or 5.6 years (CEE-Alone) and scans were conducted an average of 3 (CEE+MPA) or 1.4 years (CEE-Alone) post-trial termination. Cross-sectional analysis of MRI lesions was conducted; general linear models were fitted to assess treatment group differences using analysis of covariance. A (two-tailed) critical value of alpha = 0.05 was used. RESULTS: In women evenly matched within trials at baseline, increased lesion volumes were significantly related to age, smoking, history of cardiovascular disease, hypertension, lower post-trial global cognition scores, and increased incident cases of on- or post-trial mild cognitive impairment or probable dementia. Mean ischemic lesion volumes were slightly larger for the CEE+MPA group vs placebo, except for the basal ganglia, but the differences were not significant. Women assigned to CEE-Alone had similar mean ischemic lesion volumes compared to placebo. CONCLUSIONS: Conjugated equine estrogen-based hormone therapy was not associated with a significant increase in ischemic brain lesion volume relative to placebo. This finding was consistent within each trial and in pooled analyses across trials.

[Clinical observation on small doses Tripterygium wilfordii polyglycoside combined with methotrexate in treating rheumatoid arthritis].

OBJECTIVE: To observe the effect and adverse reaction of small doses Tripterygium wilfordii polyglycoside (TWP) combined with methotrexate (MT) in treating rheumatoid arthritis (RA). METHODS: Seventy RA patients were randomly divided into two groups, the control group (35 patients) and the TWP combined MT Group (TWPM group). Both of them were continued to use the non-steroidal anti-inflammatory drugs. The control group took MT 15 mg orally, once every week; the TWPM group took TWP 10 mg orally, 3 times a day, and MT 7.5 mg orally once every week. The clinical effect and adverse reaction after treatment were evaluated. RESULTS: The markedly effective rate in the control group and the TWPM group was 28.6% and 34.3% respectively, with no significant difference (P > 0.05). Data of symptoms and signs, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) were determined respectively with significant difference (P < 0.01). The rate of adverse reaction was 20 cases-times in the control group and 8 cases-times in the TWPM group. CONCLUSION: MT combined small doses of TWP in treating RA has better effect and less adverse reactions than un-combined MT.

Ligand-receptor binding measured by laser-scanning imaging.

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.

[The lateral abdominal island flap--the lateral intercostal neurovascular island flap].

This work studied the lateral abdominal island flap, its clinical value, transposition range and the practicability of a modified operative method. Five lateral abdominal island flaps were used in 5 patients. Four of them were for axillary radiation ulcers after radical mastectomy. One was for a sacral defect after resection of a recurrent fibrosarcoma. All the flaps obtained satisfactory results. Clinical applications revealed that the blood supply of the lateral abdominal skin was mainly from the lateral cutaneous branches of the 9th, 10th and 11th intercostal arteries, among which there were numerous anastomoses. The lateral abdominal island flap can be pedicled with any of these lateral cutaneous branches. The long pedicle of the flap provides a wide range of trnasposition from the axilla to the sacrum. As the pedicle of the flap contains the lateral cutaneous branch of the intercostal nerve, the flap can bring sensation function to the recipient area. The modified operative method of the lateral abdominal island flap is introduced.

Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.

Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to iC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to iC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to iC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to iC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.

Characterization of a 70-kDa, EBV gp350/220-binding protein on HSB-2 T cells.

EBV binds and infects HSB-2 T cells via a receptor distinct from CD21. To further study this novel EBV receptor, we expressed the first 470 amino acids of the EBV-gp350/220 using the baculovirus expression system. The recombinant gp350/220(1-470) has a m.w. of 95 kDa, reacts with anti-gp350/220 Abs, and binds CD21 in ELISA. Radiolabeled gp350/220(1-470) binds both HSB-2 and Raji cells. The gp350/220(1-470) protein also inhibits EBV binding to both HSB-2 and Raji, detected by flow cytometry. Lysates of HSB-2 cells compete with CD21 for binding to gp350/220(1-470), suggesting that the two receptors bind related epitopes on the recombinant protein. Scatchard analysis reveals that gp350/220(1-470) binds to 34,000 high affinity sites/HSB-2 cell (Kd = 0.92 x 10(-8) M) compared with the 97,000 high affinity sites bound/Raji cell (Kd = 1.78 x 10(-8) M). Utilizing a gp350/220(1-470)-affinity matrix, we identify a 70-kDa (55-kDa nonreduced) protein on the surfaces of 125I-labeled HSB-2 cells. Binding of this protein to the matrix is inhibited by anti-gp350/220 Ab 72A1. In summary, we characterize a novel EBV-binding molecule on HSB-2 cells, compare its reactivity with gp350/220 to that of CD21, and provide evidence of a gp350/220-reactive, 70-kDa protein on the surfaces of HSB-2 cells. In view of previous evidence of HSB-2 infectivity by EBV, we propose that the 70 kDa protein represents the novel EBV receptor.

Overexpression, purification, and characterization of third component of complement.

The third component of complement (C3) plays a critical role in both pathways of complement activation by interacting with numerous other complement proteins. To elucidate the molecular features of C3 that relate to the functional activities of the molecule, we expressed the cDNA of human complement component C3 in cultured insect cells using a baculovirus expression vector system derived from the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). The expression of C3 was controlled by the promoter of the polyhedrin gene and, when recombinant baculovirus infected insect cells were cultured in serum-free medium, C3 was detected at a level of 10 micrograms/ml of culture medium. Characterization of the recombinant C3 (rC3) by SDS-PAGE revealed that the C3 gene product was translated as a 188 kDa protein comprised of two chains of 115 kDa and 73 kDa analogous to the alpha and beta chains of serum-derived human C3 (sC3). An analysis of the glycosylation pattern of purified rC3 revealed that, whereas both the alpha and beta chains were glycosylated as in sC3, the proC3 moiety of rC3 also was glycosylated. When rC3 was produced in the High Five cell line of insect cells and evaluated for reactivity with a panel of anti-C3 monoclonal antibodies (MoAb), the results suggested that the conformation of the baculovirus expressed C3 was similar to that of native C3. When the rC3 was purified by anion exchange column chromatography, it was able to react with several C3-binding proteins (CR1, P and H), reconstitute C3-deficient serum and support the activation of both complement pathways thus demonstrating that a baculovirus-expressed C3 can participate in the formation of and can be cleaved by both the classical and alternative pathway convertases. Incubation of rC3 with factor I and H revealed that both C3 and proC3 are susceptible to cleavage by factor I.

Third component of trout complement. cDNA cloning and conservation of functional sites.

Of the 30 distinct complement proteins recognized to date, C3 is probably the most versatile and multifunctional molecule known, interacting with at least 20 different proteins. It plays a critical role in both pathways of complement activation and participates in phagocytic and immunoregulatory processes. Structural and functional analysis of C3 from different species, in addition to phylogenetic information, provides insights into the structural elements mediating the various functions. This study describes the cDNA cloning of one of two isoforms of the third complement component, C3-1, of rainbow trout (Salmo gairdneri) and the analysis of its functional sites. By screening a trout liver lambda gt11 library with anti-trout C3 chain-specific antibodies and polymerase chain reaction we have determined the cDNA sequence of trout C3-1. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides, corresponding to different regions of trout C3-1. C3-1 consists of 1640 amino acids with a calculated molecular mass of 181,497 Da. The sequence contains two potential N-glycosylation sites, one on each chain of C3. The deduced protein sequence showed 44.1, 43.3, 44.2, 44.9, 43.1, 43.8, 45.9, 29.9, and 33.1% amino acid identities to human, mouse rat, guinea pig, rabbit, cobra, frog, hagfish, and lamprey C3, whereas the identities to human C4, C5, and alpha 2M are 30.4, 28, and 22.9%, respectively. The trout C3 amino acid sequence shows clusters of high and low similarity to C3 from other species. In the regions of high similarity belong the C3 domains that contain the thiolester site and the properdin binding sites, whereas the regions that correspond to regions of human C3 where CR1 and CR2 bind show low amino acid sequence similarity. The deduced amino acid sequence shows that the C3 convertase cleavage site (Arg-Ser) is conserved in trout C3, whereas the factor I cleavage sites are Arg-Ala and Arg-Thr instead of Arg-Ser, which is found in the C3 of other species. Protein sequencing of the trout C3 fragments fixed on zymosan during complement activation confirmed the cleavage of trout C3 by trout C3 convertase and factor I at Arg-Ser and Arg-Thr, respectively.

Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.

Recent advances in microlymphatic surgery in China.

Development and advances in clinical and research work on limb lymphedema in China has progressed rapidly in recent years. The authors first performed successful lymphaticovenous anastomosis using the operating microscope for limb lymphedema in China in May, 1979. By 1983, surgery on 48 lymphedematous limbs in the authors' clinic had given good results in one-third of the cases. By the same year, 185 limbs with lymphedema were treated by lymphaticovenous anastomosis throughout China with excellent results achieved in 72.9% of the cases. Lymphology was investigated using animal models, intraluminal pressure, and venous graft substitution of lymphatics.

Experimental observation of transplantation of vein graft to lymphatics.

We attempted to transplant a vein graft to normal lymphatics (LVL) to investigate whether it could survive and remain joined to the lymphatics as the environment changed. A total of 152 rabbits were divided into 3 groups. Group 1 was an LVL saline-irrigated group (n = 40), with a patency rate of 52.5%. Group 2 was an LVL nonirrigated group (n = 34), with a patency rate of 29.4%. Group 3 was an LV group (n = 78), with a patency rate of 32%. All specimens were examined under either light microscope or scanning electron microscope. The data indicated that an LVL anastomosis is practical and that preanastomotic irrigation can improve the patency rate. In both the LVL and the LV groups, endothelial regeneration, originating from the adjacent endothelium, commenced within 1 week after the anastomosis, with complete healing within 3 to 4 weeks. The vein graft tended to become "lymphaticized." Thrombosis was the main cause of obstruction. The patency rate can be increased by irrigation of the ends with saline before performing the anastomosis. The graft intima must be kept intact, the nutritive blood vessels of the lymphatics must be preserved, and the flow must be artificially increased soon after the anastomosis.