[Malignant solitary fibrous tumors: a clinicopathological and molecular genetic analysis].

Objective: To explore the clinicopathological features, immunophenotype and molecular genetic characteristics of malignant solitary fibrous tumor (MSFT). Methods: Seven cases of MSFT were collected from the First Affiliated Hospital of Zhengzhou University from July 2018 to December 2020. Immunohistochemistry, RNA-based NGS and DNA-based NGS were performed. Results Among the 7 patients, there were 5 males and 2 females with a median age of 53 years (37-69 years). Two tumors located at skull base, and one in the tentorium of cerebellum, parietal occipital region, occipital area, chest and buttock respectively. The maximum diameter of the tumor was 2.5-20.0 cm. Microscopically, typical hemangiopericomatoid structures were noted; the tumor was cellular, fusiform or oval, very pleomorphic, with necrosis and high mitotic figures (>4/10 HPF). In some cases, classical solitary fibrous tumor morphology and dedifferentiated region were observed. Immunohistochemically, the tumor was positive for CD34 (6/7), STAT6 (7/7), bcl-2 (7/7), but negative for S-100 (7/7); CKpan or EMA was positive to varying degrees; mutated p53 was noted (3/7); Ki-67 positive index was more than 10%. NAB2-STAT6 gene fusion was typically detected in all the 7 cases. In 4 cases, ZNF415-FGFR1, COPG1-MET, IPO11-LRRC70_ncRNA-PLAG1 and Clorf198-CD274 (PD-L1) gene fusions were also detected. NOTCH1 mutation was found in 7 cases and TP53 mutation in 4 cases. TERT promoter mutations were not detected in all the cases. Conclusions: MSFT is rare and needs to be differentiated from many other spindle cell tumors. Especially when tumors express epithelial markers, they are easily misdiagnosed as sarcomatoid carcinoma and synovial sarcoma, etc. Immunohistochemistry and molecular detection of NAB2-STAT6 gene fusion have important diagnostic values. NOTCH1 and TP53 mutations may be associated with the progression of MSFT. Some patients have FGFR1 gene fusion and MET gene fusion, which may be potential therapeutic targets.

[Impact and clinical value of the revised 2019 Chinese HER-2 testing guidelines on the detect result evaluation of invasive breast cancer cases with equivocal HER-2 immunostaining by using fluorescence in situ hybridization].

Objective: To investigate the impact and clinical significance of the revised 2019 Chinese HER-2 testing guidelines on the detecting result evaluation of invasive breast cancers with equivocal HER-2 immunostaining by using fluorescence in situ hybridization (FISH). Methods: A total of 569 cases of invasive breast cancers with HER-2 (+ + ) immunostaining evaluated according to the immunohistochemistry (IHC) guidelines of 2014 edition and 2019 edition from May to November 2019 were collected and further detected by FISH. The results of HER-2/CEPl7 double probe were respectively interpreted according to both the 2014 and 2019 Chinese HER-2 testing guidelines and the results were compared. Results: According to the 2014 guidelines, the number of HER-2 positive, equivocal and negative cases were 139 (24.43%), 67 (11.78%), and 363 (63.80%), respectively. Whereas according to the 2019 guidelines, 115 cases (20.21%) were the first group, 9 cases (1.58%) were the second group, 15 cases (2.64%) were the third group, 67 cases (11.78%) were the fourth group, and 363 cases were (63.80%) the fifth group, of which 130 cases (22.85%) were positive and 439 cases (77.15%) were negative by FISH detecting. Compared with the guideline of 2014 edition, the HER-2 positive rate of FISH detection reduced from 24.43% (139/569) to 22.85% (130/559) according to the application of the guideline of 2019 edition, but the difference was not statistically significant (P=0.567), while the negative rate increased from 63.80% (363/569) to 77.15% (439/569), with a statistically significant difference (P<0.05). Forty-three cases with incomplete weak to medium intensity of IHC membrane staining which were HER-2 (+ + ) according to 2014 guideline were changed to IHC (+ ) on the basis of the 2019 guideline. According to the FISH guideline of 2014 edition, 1 case (2.33%) was positive, 6 cases (13.95%) was equivocal and 36 cases (83.72%) was negative, while according to the 2019 FISH guideline, all of the 43 cases were negative. Conclusions: According to the guideline of 2019 edition, a proportion of cases changes from HER-2 (+ + ) to (+ ), and the HER-2 positive rate of FISH test decreases slightly, the negative rate increases, the equivocal result is eliminated, which provides a definite reference for screening patients who will be benefited from the targeted treatment of HER-2.

[Establishment and validation of prognostic risk score model for esophageal squamous cell carcinoma based on immune related genes].

Objective: To find the biomarkers that accurately predict the survival of patients with esophageal squamous cell carcinoma (ESCC). Methods: The immune related genes that were significantly related to the overall survival (OS) of patients with ESCC were screened from The Cancer Genome Atlas (TCGA) database to construct a prognostic risk score model. The prognoses of the high-risk and low-risk groups were compared by Kaplan-Meier method. The accuracy of the model was evaluated by the receiver operating characteristic (ROC) curve. Tumor tissue samples of 83 patients with pathological diagnosis of ESCC were collected from Anyang Cancer Hospital for external verification. Cox regression analysis was used to comprehensively evaluate the effects of prognostic risk score and various clinical characteristics on OS of patients with ESCC. Results: Seven immune-related genes that were significantly related to survival prognosis were selected from the TCGA database and included in the prognostic risk score model, which were S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2. The 1- and 2-year survival rates of the low-risk group (40 cases) were 94.3% and 82.5%, respectively, while those of the high-risk group (40 cases) were 75.9% and 32.9%, respectively.The prognosis of the high-risk group was worse than that of the low-risk group (P<0.001). The 83 external validation samples obtained consistent results by using the prognostic risk score model. The prognostic risk score was positively correlated with the content of CD4(+) T lymphocytes in ESCC (r(s)=0.259, P=0.020), but not correlated with the content of B lymphocytes, CD8(+) T lymphocytes, neutrophils, macrophages or dendritic cells (P>0.05). Conclusions: S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2 were risk genes significantly associated with OS of patients with ESCC. The prognostic risk score was an independent prognostic factor for the OS of patients with ESCC, and it was correlated with the content of CD4(+) T lymphocytes in ESCC tissue.

[Nodal lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia: a clinicopathological and prognostical study].

Objective: To study the clinicopathological features and prognosis of nodal lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia (n-LPL/WM). Methods: A total of 19 cases of n-LPL/WM were collected from May 2009 to January 2020 at First Affiliated Hospital of Zhengzhou University. The clinicopathologic features, immunophenotype, Ig gene rearrangement (BIOMED-2), MYD88 L265P mutation status (by Sanger sequencing) and follow-up data (by telephone) were analyzed. Results: There were 15 males and 4 females with a median age of 61 years (range 43 to 82 years). There were 14 WM and five LPL. The most common symptoms were weakness, fatigue (9/19) and B symptoms (11/19). Majority of the patients (16/18) presented with systemic multiple lymphadenopathies. Eighteen patients presented at advanced stages (Ⅲ/Ⅳ stage). Serum M protein status was IgM (15 cases), IgG (1 case), IgA (1 case) and no-secretory type (2 cases). Seventeen patients had bone marrow involvement. Morphologically, all 19 cases were divided into two groups: typical group (9 cases) or atypical group (10 cases). In the typical group, the structures of the lymph nodes were preserved; the neoplastic cells were predominantly plasmacytoid lymphocytes or mixed small lymphocytes, plasmacytoid lymphocytes and plasma cells, without proliferation of FDC network and follicular implantation. In the atypical group, the tumor showed effaced nodal architecture (5 cases), mainly proliferation of small lymphocytes (6 cases), FDC proliferation and/or follicular implantation (6 cases), marginal zone B cell differentiation (4 cases) and diffuse amyloidosis (1 case). Hemosiderin deposition (19 cases), infiltration of fatty tissue (19 cases) and interstitial sclerosis (9 cases) were commonly seen in both groups. Immunohistochemically, the neoplastic B cells expressed CD20 and CD79α, and the neoplastic plasma cells were positive for CD38, CD138 and MUM-1; eight cases showed light chain restriction; of the seven detected cases, five expressed IgM and the other two expressed IgG and IgA respectively; four cases expressed CD23 weakly, Ki-67 index was 10%-30%. MYD88 L265P mutation was seen in 18/18 cases. There was no significant difference in clinicopathologic features and prognosis between the two groups (P>0.05). The median follow-up time was 61 months, 11 patients were alive, while eight died; the 5-year survival rate was 21.1%. Conclusions: n-LPL/WM is rare, but patients usually present in advanced stages. It is easily confused with other small B-cell lymphomas with plasma cell differentiation, especially basing on morphologic features alone; thus the accurate diagnosis of n-LPL/WM requires a combination of clinical features, serum M protein, immunohistochemistry, bone marrow morphology,flow cytometry and MYD88 L265P mutation status etc. The prognosis of n-LPL/WM may be not very good, and further studies with more cases are needed.

[Clinicopathological features and MYD88 L265P mutation status of intravascular large B cell lymphoma].

Objective: To study the clinicopathologic features and MYD88 L265P mutation status of intravascular large B cell lymphoma (IVLBCL). Methods: Fourteen cases of IVLBCLs were diagnosed from March 2014 to December 2019 at the First Affiliated Hospital of Zhengzhou University. The clinicopathologic features and prognosis were analyzed. Epstein-Barr virus encoded RNAs and MYD88 L265P mutation status were detected using in situ hybridization and Sanger sequencing, respectively. The follow-up data were obtained by telephone interview. Results: There were 6 males and 8 females with a median age of 62 years (range: 48-73 years). The involved anatomic locations were demonstrated by positron emission tomography-computed tomography, including adrenal gland (7/14), bone (6/14), central nerve system (4/14), skin (3/14), female reproductive system (3/14), local lymph nodes (3/14), prostate (2/14), liver and spleen (2/14), sphenoid sinus (1/14), penis (1/14), bladder (1/14), and right lung (1/14). Fever was the most common symptom (7/14), followed by neurologic symptoms and lower abdominal pain (2/14 each). The reminder symptoms included rash with edema, legs weakness and numbness, or postmenopausal bleeding (1/14 each). Eleven cases were at Lugano stage Ⅳ. Four cases were associated with the hemophagocytic syndrome, while 6 cases with bone marrow involved. Microscopically, the tumor cells were generally concentrated within the small-to-medium vascular lumens or sinusoids; they had centroblast-like appearance and showed large round or oval nuclei with slightly irregularities, coarse chromatin and 1-3 distinct nucleoli. One exception was the one case with an embryoid nuclei, reminiscent of anaplastic large cell lymphoma. The mitosis was not uncommon. Extravascular neoplastic cells were seen in two cases. The neutrophils could be appreciable in most of the cases (10/14). Immunophenotyping showed that CD20 and CD79α were diffusely and strongly positive in 14 cases; 12 cases were classified as the non-GCB subtype; 6 out of the 11 cases were double expressor lymphoma; 7 out of the 12 cases were CD5-positive. Twelve cases were EBER negative. The MYD88 L265P mutation was detected in 1 case (1/10). The duration of the follow-up ranged from 0.5 to 24.0 months, and 11 patients survived and 3 died. Conclusions: IVLBCL is rare. The most common type of IVLBCL in China is Asian type with scant tumor cells. Combination of clinical and immunohistochemical features can avoid most, if not all, misdiagnoses and missed diagnoses. Some IVLBCL cases may harbor the MYD88 L265P mutation, but the prevalence of MYD88 L265P mutation in the population still warrants additional studies.

[Gastrointestinal glomus tumors: a clinicopathological analysis of fifteen cases].

Objective: To investigate the clinicopathological features, diagnosis and differential diagnosis of gastrointestinal glomus tumors (GIGT). Methods: Totally 15 cases of GIGT were collected at the First Affiliated Hospital, Zhengzhou University, from January 2011 to June 2018. The clinicopathological features, immunophenotype, BRAF V600E mutation and prognosis were retrospectively analyzed. Results: The 15 patients' age ranged from 37 to 59 years(median 49 years, mean 50 years). Eleven patients presented with intermittent abdominal pain and distention, three showed antral space-occupying lesions at physical examination, and one had abdominal pain accompanied by fecal blood. Fourteen tumors were located in the stomach, and one was in the ileum. Imaging showed the gastric glomus tumors were located in the submucosal layer with obvious enhancement in the arterial phase, and the ileum glomus tumor involved the whole layer of intestinal wall causing luminal obstruction. The maximum diameters of the tumors ranged from 1.5 to 3.0 cm (mean 2.3 cm). Grossly, the gastric glomus tumors were solid. Microscopically, the gastric glomus tumors were mostly located in the muscularispropria layer and were vascular. The tumor boundary was distinct but without capsule formation. The tumor cells were round or oval, and showed perivascular hemangiopericytoma-like or solid nest-like structures. The tumor cells were mildly pleomorphic, with rare mitosis and no necrosis. Two tumors had focal calcification, two showed mucosal invasion, two showed vascular invasion and five showed perineural invasion. The ileum glomus tumor was cellular, with prominent cellular atypia, and the mitotic count in hot spots was about 5-6/HPF. Immunohistochemistry showed that SMA and collage Ⅳ were strongly expressed in all the tumor cells; caldesmon and calponin were moderately expressed in some regions, and syn was weakly expressed in 12 cases. The Ki-67 proliferation index in the gastric glomus tumors ranged from 1% to 30% (mean 6%); and that in the ileum glomus tumor was about 70%. BRAF V600E mutations were not detected in any of 15 GIGTs. All patients did not receive radiotherapy or chemotherapy post operatively. Thirteen patients were followed up by telephone for 18-90 months (mean 42 months). Twelve patients with gastric glomus tumors survived without recurrence and metastasis, and the patient with ileum glomus tumor had liver metastasis 15 months after operation. Conclusions: Glomus tumors is a rare mesenchymal tumor of the gastrointestinal tract. It should be differentiated from gastrointestinal stromal tumors, neuroendocrine tumor, leiomyoma, solitary fibrous tumor and paraganglioma. Most GIGTs are benign and have good prognosis. More experience is needed to understand the biologic behavior and prognostication of GIGTs.

Effect of RhoC silencing on multiple myeloma xenografts and angiogenesis in nude mice.

In this study, we investigated the expression of RhoC in the multiple myeloma (MM) cell line RPMI- 8226, as well as the effects of silencing RhoC on the growth of tumor xenografts and tumor-induced angiogenesis in nude mice with MM. For this purpose, we transduced RPMI-8226 cells with lentiviral particles overexpressing short hairpin RNAs (shRNA) targeting RhoC. Tumor xenografts were generated by subcutaneously injecting nude mice with RPMI-8226 cells overexpressing control shRNA [negative control (NC) group] or the RhoC shRNA [the experimental (S) group], respectively. RhoC protein and mRNA levels in the tumor xenografts were measured. Nude mice were also subcutaneously inoculated with Matrigel mixed with vascular endothelial growth factor, and CD31 and KI67 levels in the tumor xenografts were measured by immunohistochemistry. Similarly, we assessed tumor xenograft growth and angiogenesis in Matrigel implants in the mice of both groups. We found that RhoC levels, microvessel density, and CD31 labeling index were more reduced in the S group than in the NC group. However, there was no significant difference in the size of tumor xenografts between the 2 groups. The number of new vessels and the neovascular length in the Matrigel implants were significantly lower in the S group than in the NC group. Therefore, we concluded that RhoC expression in myeloma xenografts has important effects on the induction of angiogenesis.

First Report of Alternaria heveae Causing Black Leaf Spot of Rubber Tree in China.

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop of tropical areas for natural rubber production. In October 2013, foliar spots (0.1 to 0.4 mm in diameter), black surrounded by a yellow halo, and with lesions slightly sunken were observed on the rubber tree leaf in a growing area in Heikou County of Yunnan Province. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on PDA plates, and incubated at 28°C with alternating day/night cycles of light. The pathogen was observed growing out of many of the leaf pieces, and produced abundant conidia. Colonies 6.1 cm in diameter developed on potato carrot agar (PCA) after 7 days, with well-defined concentric rings of growth. Colonies on PCA were composed of fine, dark, radiating, surface and subsurface hyphae. Conidia produced in PCA culture were mostly solitary or in short chains of 2 to 5 spores, long ovoid to clavate, and light brown, 40 to 81.25 × 8 to 20 µm (200 colonies were measured), with 3 to 6 transverse septa and 0 to 2 longitudinal or oblique septa. Morphological characteristics were similar to those described for Alternaria heveae (3,4). A disease of rubber tree caused by Alternaria sp. had been reported in Mexico in 1947 (2). DNA of Ah01HK13 isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers was completed. From the BLAST analysis, the sequence of Ah01HK13 (GenBank Accession No. KF953884), had 97% similarity to A. dauci, 96% identical to A. macrospora (AY154701.1 and DQ156342.1, respectively), indicating the pathogen belonged to Alternaria genus. According to morphological characteristics, this pathogen was identified as A. heveae. Pathogenicity of representative isolate, Ah01HK13 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage and inoculated by spraying spore suspension (concentration = 104 conidia/ml) to the copper-colored leaves until drops were equally distributed on it using manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags. The plastic bags were removed after 2 days post-inoculation (dpi) and monitored daily for symptom development (1). The experiment was repeated three times. The typical 0.1 to 0.4 mm black leaf spots were observed 7 dpi. No symptoms were observed on control plants. A fungus with the same colony and conidial morphology as A. heveae were re-isolated from leaf lesions on inoculated rubber plants, but not from asymptomatic leaves of control plants, fulfilling Koch's postulates. Based on these results, the disease was identified as black spot of rubber tree caused by A. heveae. To our knowledge, this is the first report of A. heveae on rubber tree in China. References: (1) Z. Y. Cai et al. Microbiol Res. 168:340, 2013. (2) W. J. Martin. Plant Dis. Rep. 31:155, 1947. (3) E. G. Simmons. Mycotaxon 50:262, 1994. (4) T. Y. Zhang. Page 111 in: Flora Fungorum Sinicorum: Alternaria, Science Press, Beijing, 2003.

Role of CD86 (B7-2) in triggering of antigen-specific IgE antibody response by lipopolysaccharide.

The role of CD86 in triggering of ascaris extract-specific IgE antibody response by lipopolysaccharide was studied. The simultaneous administration of anti-CD86 antibody with ascaris extract and lipopolysaccharide prevented the production of IgE antibody response to ascaris extract. CD86+ cells were detected in peritoneal cavities and spleens of mice injected intraperitoneally with lipopolysaccharide. CD86+ cells appeared in peritoneal cavities and spleens eight hours after lipopolysaccharide injection, and they were detectable for a week. CD86+ cells in peritoneal cavities and spleens were mainly surface Ig-positive B-cells and some Ig-negative cells. It was suggested that lipopolysaccharide induced the expression of CD86 mainly on B-cells, and that CD86+ cells induced by lipopolysaccharide injection might play an important role as antigen-presenting cells on triggering of ascaris extract-specific IgE antibody response by lipopolysaccharide.

Augmentation of lipopolysaccharide-induced thymocyte apoptosis by interferon-gamma.

The role of interferon (IFN)-gamma on thymocyte apoptosis in response to lipopolysaccharide (LPS) was investigated. The administration of LPS into mice induced marked apoptosis of thymocytes in vivo, but the simultaneous injection of anti-IFN-gamma antibody with LPS completely prevented thymocyte apoptosis. Pretreatment of mice with IFN-gamma markedly enhanced LPS-induced thymocyte apoptosis. Thymocyte apoptosis augmented by IFN-gamma occurred in the thymic cortex, and target cells undergoing apoptosis were CD4+8+ immature thymocytes. IFN-gamma itself did not induce thymocyte apoptosis in vivo and in vitro. IFN-gamma exhibited no synergistic action with effector molecules, such as tumor necrosis factor (TNF)-alpha and glucocorticoids. Further, it was shown that IFN-gamma did not enhance the susceptibility of thymocytes to apoptosis. Pretreatment of mice with IFN-gamma significantly augmented the serum TNF-alpha level and the serum cortisol level in response to LPS. Therefore, we suggest that IFN-gamma might augment LPS-induced thymocyte apoptosis through elevating serum TNF-alpha and cortisol levels.

Marked reduction of mouse peritoneal CD5+ B cells by intraperitoneal administration of lipopolysaccharide.

Intraperitoneal administration of lipopolysaccharide to mice induced a marked reduction of CD5+ B cells in the peritoneal cavity. The reduction was not induced by intravenous, subcutaneous, or oral administration of lipopolysaccharide. The reduction continued for about 10 days after the injection, and the CD5+ B-cell count recovered to the normal state about 14 days after the injection. The reduction of peritoneal CD5+ B cells might be caused by apoptotic cell death. Injection of lipopolysaccharide did not result in production of antibody to lipopolysaccharide. On the other hand, intraperitoneal injection of heat-killed bacteria did not induce a reduction of peritoneal CD5+ B cells and elicited the definite production of antibody to lipopolysaccharide.

Apoptotic cell death of vascular endothelial cells and renal tubular cells in the generalized Shwartzman reaction.

The participation of apoptotic cell in the generalized Shwartzman reaction was examined. The generalized Shwartzman reaction was induced in mice by two consecutive injections of lipopolysaccharide. Vascular endothelial cells in various organs of those mice were stained positively by the in situ specific labeling of fragmented DNA. Renal tubules were also stained focally. It was suggested that apoptotic cell death might participate in the development of vascular endothelial cell damage and acute tubular necrosis in the generalized Shwartzman reaction. Simultaneous administration of anti-gamma-interferon antibody in the preparative injection of lipopolysaccharide completely blocked apoptosis of vascular endothelial cells. Priming with recombinant gamma-interferon instead of lipopolysaccharide could produce apoptosis of vascular endothelial cells. It was suggested that gamma-interferon might play a critical role on sensitization of endothelial cells for apoptosis.

Differential release of smooth-type lipopolysaccharide from Pseudomonas aeruginosa treated with carbapenem antibiotics and its relation to production of tumor necrosis factor alpha and nitric oxide.

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.

Lipopolysaccharide induces apoptotic cell death of B memory cells and regulates B cell memory in antigen-nonspecific manner.

Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig+ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.

Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.

The apoptotic cell death induced in D-galactosamine-sensitized mice by administration of lipopolysaccharide was characterized. Administration of lipopolysaccharide caused apoptotic cell death in livers of D-galactosamine-sensitized mice. Apoptotic cells were also detected in the kidney, thymus, spleen, and lymph node. Severe hepatic apoptosis in D-galactosamine-sensitized mice was reproduced by transfer of the sera from mice injected with D-galactosamine and lipopolysaccharide. The hepatocyte apoptosis induced by lipopolysaccharide was completely prevented by an anti-tumor necrosis factor alpha antibody but not by an anti-gamma interferon antibody. Administration of recombinant tumor necrosis factor into D-galactosamine-sensitized mice also caused hepatocyte apoptosis. Lipopolysaccharide-induced hepatocyte apoptosis in D-galactosamine-sensitized mice did not seem to be mediated by Fas antigen. It was suggested that lipopolysaccharide- induced hepatic injury and failure in D-galactosamine-sensitized mice was due to the apoptotic cell death of hepatocytes caused by tumor necrosis factor alpha released in the circulation.

Role of tumor necrosis factor-alpha and glucocorticoid on lipopolysaccharide (LPS)-induced apoptosis of thymocytes.

Administration of bacterial lipopolysaccharide (LPS) into mice markedly induced the apoptosis of CD4+8+ thymocytes. The injection of anti-tumor necrosis factor (TNF)-alpha antibody or RU38486, a glucocorticoid receptor antagonist, into mice definitely inhibited LPS-induced apoptosis of thymocytes. Addition of the sera 1 h after injection of LPS into in vitro cultures of thymocytes caused thymocyte apoptosis. It was also prevented by either anti-TNF-alpha antibody or RU38486. Further, recombinant TNF-alpha and hydrocortisone collaborated in induction of the thymocyte apoptosis in vitro. The in vivo phenomenon of LPS-induced apoptosis of thymocytes was reproducible by the in vitro experimental system. It was therefore suggested that both TNF-alpha and glucocorticoid participate and collaborate as effector molecules in LPS-induced apoptosis of thymocytes.

Binding of mannose-binding protein to Klebsiella O3 lipopolysaccharide possessing the mannose homopolysaccharide as the O-specific polysaccharide and its relation to complement activation.

Lipopolysaccharide from Klebsiella pneumoniae O3, which possesses the mannose homopolysaccharide as the O-specific polysaccharide, exhibits an extraordinarily high ability to activate the human complement system. We isolated the mannose-binding protein with a Klebsiella O3 lipopolysaccharide affinity column. The protein isolated had a molecular mass of much higher than 200 kDa, and it consisted of subunits with an apparent molecular mass of 32 kDa. The NH2-terminal sequence of the 32-kDa subunits was completely consistent with a part of the amino acid sequence of human serum mannose-binding protein. In immunoblotting, an anti-mannose-binding protein monoclonal antibody was definitely reactive with the isolated protein with the higher molecular mass. The protein isolated was bound exclusively to lipopolysaccharides possessing the mannose homopolysaccharide, not to lipopolysaccharide possessing the heteropolysaccharides. Klebsiella O3 lipopolysaccharide did not exhibit a high anticomplement activity in the serum from which the mannose-binding protein was depleted. It was concluded that the serum factor that bound to Klebsiella O3 lipopolysaccharide may be mannose-binding protein and that it may play a crucial role in the strong complement activation by Klebsiella O3 lipopolysaccharide.

Strong adjuvant action of Klebsiella O3 lipopolysaccharide and its inhibition of systemic anaphylaxis.

Immunization with lipopolysaccharide from Klebsiella O3 as an immunological adjuvant did not cause the death of mice in systemic anaphylaxis to bovine serum albumin. On the other hand, most mice immunized with lipopolysaccharide from Escherichia coli O111, Klebsiella O4 and Salmonella minnesota did die. Klebsiella O3 lipopolysaccharide enhanced IgM and IgG antibody response to BSA more markedly than Escherichia coli O111 lipopolysaccharide, while it affected the production of IgE antibody only slightly. therefore, it is suggested that the inhibition of systemic anaphylaxis by Klebsiella O3 lipopolysaccharide adjuvant might be related to its strong adjuvant action on IgM and IgG class antibody production, and that high levels of circulating IgM and IgG antibodies might act as blocking antibodies in the development of IgE-mediated systemic anaphylaxis.

In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.