[The changes of perioperative immunity index in patients with breast cancer and its clinical significance].

Objective: To investigate the changes of perioperative immune index in patients with breast cancer and its clinical significance. Methods: Th1 cells, Th2 cells, Th1/Th2 ratio and regulatory T cells (Treg) were detected in peripheral blood of 103 patients with primary breast cancer and 116 patients with breast fibroma before surgery and on the 1st, 3rd and 5th day following operation. The relationship of changes in T lymphocyte subsets and clinicopathological characteristics, as well as tumor-free survival of breast cancer patients, was analyzed. Results: The levels of Th1 cells in breast cancer group on the 1st, 3rd and 5th day following operation were (12.20±0.45)%, (13.89±0.47)%, (14.04±0.49)%, which were significantly lower than those before operation [(15.82 + 0.51)%, all P<0.05 ]. Treg cells, however, with the number of (3.82±0.13)%, (3.25±0.11)%, (2.95 ±0.11)%, were remarkably higher than those before operation [(2.53 ±0.11)%, all P<0.05]. With respect to breast fibroma patients, there was no significant difference compared with those before operation of Th1 cells, Th2 cells and Treg cells (all P>0.05). The changes of Th1 cells were associated with the degree of differentiation, T stage, N stage, TNM stage, HER-2 status and Ki-67 (all P<0.05). Treg cells were related to T stage, N stage and HER-2 status (all P<0.05). Tumor-free survival in the Th1-cell-increasing group was significantly better than that in the Th1-cell-decreasing group (P=0.045), while cell-decreasing group of Treg showed the improved outcomes (P=0.012). Conclusions: The levels of Th1 cells and Treg cells are important indicators of cellular immune function in patients with breast cancer. Moreover, the perioperative changes of Th1 cells and Treg cells are associated with the size of tumors, pathological parameters, clinical stages and tumor-free survival outcomes.

[Etiological analysis and establishment of a discriminant model for lower respiratory tract infections in hospitalized patients].

Objective: To analyze the pathogens of lower respiratory tract infection(LRTI) including bacterial, viral and mixed infection, and to establish a discriminant model based on clinical features in order to predict the pathogens. Methods: A total of 243 hospitalized patients with lower respiratory tract infections were enrolled in Fujian Provincial Hospital from April 2012 to September 2015. The clinical data and airway (sputum and/or bronchoalveolar lavage) samples were collected. Microbes were identified by traditional culture (for bacteria), loop-mediated isothermal amplification(LAMP) and gene sequencing (for bacteria and atypical pathogen), or Real-time quantitative polymerase chain reaction (Real-time PCR)for viruses. Finally, a discriminant model was established by using the discriminant analysis methods to help to predict bacterial, viral and mixed infections. Results: Pathogens were detected in 53.9% (131/243) of the 243 cases.Bacteria accounted for 23.5%(57/243, of which 17 cases with the virus, 1 case with Mycoplasma pneumoniae and virus), mainly Pseudomonas Aeruginosa and Klebsiella Pneumonia. Atypical pathogens for 4.9% (12/243, of which 3 cases with the virus, 1 case of bacteria and viruses), all were mycoplasma pneumonia. Viruses for 34.6% (84/243, of which 17 cases of bacteria, 3 cases with Mycoplasma pneumoniae, 1 case with Mycoplasma pneumoniae and bacteria) of the cases, mainly Influenza A virus and Human Cytomegalovirus, and other virus like adenovirus, human parainfluenza virus, respiratory syncytial virus, human metapneumovirus, human boca virus were also detected fewly. Seven parameters including mental status, using antibiotics prior to admission, complications, abnormal breath sounds, neutrophil alkaline phosphatase (NAP) score, pneumonia severity index (PSI) score and CRUB-65 score were enrolled after univariate analysis, and discriminant analysis was used to establish the discriminant model by applying the identified pathogens as the dependent variable. The total positive predictive value was 64.7%(77/119), with 66.7% for bacterial infection, 78.0% for viral infection and 33.3% for the mixed infection. Conclusions: The mostly detected pathogens were Pseudomonas aeruginosa, atypitcal pathogens, Klebsiella pneumoniae, influenza A virus and human cytomegalovirus in hospitalized patients with LRTI in this hospital. The discriminant diagnostic model established by clinical features may contribute to predict the pathogens of LRTI.

Clinical characteristics and biomarkers of breast cancer associated with choline concentration measured by 1H MRS.

This study investigated the association between the total choline (tCho) concentration and the clinical characteristics and biomarker status of breast cancer. Sixty-two patients with breast cancer, 1.5 cm or larger in size on MR images, were studied. The tCho concentration was correlated with the MRI features, contrast enhancement kinetics, clinical variables and biomarkers. Pairwise two-tailed Spearman's nonparametric test was used for statistical analysis. The tCho concentration was higher in high-grade than moderate-/low-grade tumors (p = 0.04) and in tumors with higher K(trans) and k(ep) (p < 0.001 for both). The association of tCho concentration with age (p = 0.05) and triple negative biomarker (p = 0.09) approached significance. tCho was not detected in 17 patients, including 15 with invasive ductal cancer and two with infiltrating lobular cancer. Fifteen of the 17 patients had moderate- to low-grade cancers, and 11 had human epidermal growth factor-2-negative cancer, suggesting that these two factors might lead to false-negative choline. Higher tCho concentration in high-grade tumors and tumors with higher K(trans) and k(ep) indicates that choline is associated with cell proliferation and tumor angiogenesis. The higher choline level in younger women may be caused by their more aggressive tumor type. The results presented here may aid in the better interpretation of (1)H MRS for the diagnosis of breast lesions.

[LPS and PMA induced PKC-alpha and PKC-epsilon activation and translocation in murine peritoneal macrophages].

Suppressor macrophages induced by the continuous invasion of tumor cells and parasites, which can acquire an ability in vitro to kill or inhibit tumor cells and inhibit the activity of T, B lymphocytes and NK cells, have been indicated. We have developed a procedure previously to modulate the suppressor macrophages by bacterial lipopolysaccharide (LPS). The modulated macrophages remained and even enhanced the ability to inhibit tumor growth and to up-regulate or enhance the activities of T, B lymphocytes and NK cells in vitro. However, the mechanisms of macrophage modulation by LPS are unknown. This investigation was designed to analyze the regulation of PKC activity and to characterize the isoforms of PKC during macrophage modulation by using Western blot and endogenous substrate phosphorylation (PKC-DESP). In rest cells, PKC-beta was found to be the most abundant isoform in macrophages; and PKC-alpha, beta was found predominantly in the cytosol. Using PMA as a positive control, we found that the immuno-modulator agent--LPS triggered the physical translocation from the cytosol onto the membrane of PKC-alpha and PKC-epsilon, but PKC-beta (beta I or beta II) was difficult to detect. The analysis of PKC-DESP showed a pattern with a time course similar to that observed with Western blot. We observed that LPS and PMA increase the level of phosphorylation of 55 kDa and 74 kDa proteins with a corresponding decrease in the cytosolic proteins. It suggests that the translocation of PKC-alpha and PKC-epsilon, may be important events involving in the PKC-pathway by LPS-mediated modulation in suppressor macrophages.

[Expression of murine interleukin 12(mIL-12) in insect cells].

Since human IL-12 is species-specific in its functions and elicits little biological responses from mouse lymphocytes, it is necessary to express recombinant murine IL-12 for the usage in studying the effects of this cytokine in various rodent models. Thereby, we can investigate the role of IL-12 in immune response in vivo and evaluate its potential clinical utility. Thus, we firstly constructed two expression vectors, pVL1393-mp40 and pVL1393-mp35. They were used to co-transfect the insect cells(Sf9) separately with linearized polyhedrosis virus genomic DNA. Two kinds of recombinant viruses AcNPV-mp40 and AcNPV-mp35 were visually screened out, and mp40 and mp35 were co-expressed in the insect cells co-infected by AcNPV-mp40 and AcNPV-mp35. The results of real-time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the recombinant mIL-12 was expressed successfully in the insect cells. The molecular weights of recombinant mp40 and mp35 were 40 KDa and 22 KDa on SDS-PAGE under reducing conditions, respectively. The apparent molecular weight of recombinant mIL-12 is 80 KDa under non-reducing conditions of Western blot. Biological activity of the recombinant product was detected in conditional medium using antibody-capture bioassay. The expression level of recombinant mIL-12 was about 10-15 micrograms/10(6) cells, as compared with the calibration curve of mIL-12.

[Studies on cell signaling immunomodulated murine peritoneal suppressor macrophages: LPS and PMA mediate the activation of RAF-1, MAPK p44 and MAPK p42 and p38 MAPK].

Monocytes-macrophages which serve as host immune cells to kill pathogens can often be "activated" after exposing to viruses, bacteria, cytokines as well as chemical substances, However, it is paradoxical that highly activated macrophages can be induced to become the suppressor ones by live microbes, microbial products, tumor, and autoimmune disease, although the mechanism remains unknown. Our previous experimental studies have shown that immuno-suppressor activities of suppressor macrophages on T, B and NK cells can be prevented by the treatment with LPS or supernatant in vitro from mitogen-stimulated lymphocytes, while, at the same time, the tumoricidal activities of those macrophages can be kept or even enhanced following the same treatment. This phenomenon was then termed as "immune modulation" For the understanding of its mechanism, we are now undertaking signal transduction in modulated macrophages. Since mitogen-activated protein kinase (MAPK) is an integration point of different signal transduction pathways, its cascade and regulation of activation are being investigated extensively by the assay of electrophoresis mobility shift. Recent results suggested that interaction of ligand-receptor triggers protein tyrosine kinase(PTK) activation leading to Ras-GTP binding with Raf-1 to phosphorylate MAPK kinase (MAPKK), the specific activator of MAPK. It is reported that PKC-alpha can directly phosphorylate or activate Raf-1 in NIH3 T3 cells. Raf-1 (74 KDa), with an intrinsic serine (Ser)-threonine (The) kinase activity, becomes hyperphosphorylated after activation which can be followed by gel mobility shift test. It has also been shown that a variety of extracellular factors stimulate a pair of MAPK p44 and MAPK p42 of MAPK family members. A significant property of activation of ERK 1 and ERK 2 is the requirement for the phosphorylation of both Thr-183 and Tyr-185 (at TEY motif) within in its protein kinase subdomain VIII. More recently, two other MAPK subtypes, p38 MAPK (mammalian equivalents of HOG1 in yeast) and JNK MAPK have been discovered. The requirement for activation of p38 MAPK for both Thr-180 and Tyr-182 (at TGY motif) has been shown. p38 MAPK is important in certain transcriptional regulatory pathways, since it can phosphorylate the following transcriptional factors: 1) Elk at Ser 383/389 for binding with SRE motif; 2). ATF 2 at Ser 69/71, forming a complex with Myc for DNA binding at CRE motif; 3) Max at Ser-62 to combine DNA of E-Box motif. p38 MAPK can be activated by LPS, inflammatory cytokines, such as TNF and IL-1, osmolarity. To examine the possibility that whether activation of Raf-1 and ERK 1, ERK2 and p38 MAPK can be regulated directly or/and differently by PKC and PKA pathways, herbimycin A (Ki = 0.9 mumol/L), a potent PTK inhibitor (J. Immunol. 155:3944-4003, 1995) at 2 mumol/L concentration was utilized to block Ras/Raf-1/MAPK cascade. After pre-incubation of macrophages with herbimycin A for 30 min or 90 min, cells were treated with LPS (10 micrograms/ml) and PMA (100 nmol/L) for 15 min. No inhibition of phosphorylation of Raf-1, MAPK p44 and MAPK p42 in response to LPS and PMA was observed (Fig. 1 and 3). However, forskolin, a cAMP inducer for protein kinase A (PKA) activation, inhibited the phosphorylation of LPS- and PMA-stimulated Raf-1, MAPK p44 and MAPK p42 (Fig. 2 and 4). Similarly, in agreement with a very recent report from David, M et al in NIH, in which they indicated that forskolin (30 mumol/L) inhibited IFN-beta-stimulated ERK activity by U 266 cells (J. Biol. Chem. 271: 4585-4588 1996), we found that the levels of phosphorylations of Raf-1 and ERK1 and ERK2 were declined when forskolin (30 mumol/L) was added to macrophages for 20 min at 37 degrees C prior to the stimulation by LPS and PMA. Interestingly, under the same condition, forskolin (30 mumol/L) stimulated the phosphorylation of LPS- and PMA-triggered p38 MAPK of murine peritoneal suppressor macrophages, suggesting that activatio