[Pathological characteristics of megakaryocytes in myeloproliferative neoplasms and their correlation with driver gene mutations].

Objective: To investigate the pathological characteristics of megakaryocytes in myeloproliferative neoplasms(MPN)and their correlations with driver gene mutations. Methods: Trephine specimens administered for 160 patients with MPN from February 2012 to October 2017 were reevaluated according to the World Health Organization(WHO)'s(2016)diagnostic criteria. Results: This cohort of patients included 72(45.0%)men, with the median age of 59(range, 13-87)years, comprising 39 with polycythemia vera(PV), 33 with essential thrombocythemia(ET), 37 with prefibrotic/early-primary myelofibrosis(pre-PMF), 37 with overt PMF, 1 with post-ET MF, 2 with post-PV MF, and 11 with MPN-unclassifiable(MPN-U)after the re-diagnosis. With PV, ET, pre-PMF, and overt PMF changes, proportions of dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes gradually increased, whereas erythropoiesis gradually decreased. Proportions of reticulin, collagen, and osteosclerosis grades of ≥1 also increased. Dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes were negatively correlated with erythropoiesis and positively correlated with granulopoiesis and fibrosis. In patients with pre- and overt PMF, dense clusters and naked nuclei of megakaryocytes were positively correlated with fibrosis. Patients with JAK2V617F MPN had significantly increased erythropoiesis(P=0.022). Patients with CALR-mutated MPN were characterized by increased loose and dense clusters; paratrabecular distribution and naked nuclei of megakaryocytes(P=0.055, P=0.002, P=0.018, P=0.008); and increased reticulin, collagen, and osteosclerosis(P=0.003, P<0.001, P=0.001). In patients with pre- and overt PMF, patients with JAK2V617F had increased cellularity(P=0.037). CALR-mutated patients had increased dense clusters and giant sizes of megakaryocytes, collagen, and osteosclerosis(P=0.055, P=0.059, P=0.011, P=0.046). Conclusion: Megakaryocytes showed abnormal MPN morphology and distribution, which were related to fibrosis. CALR mutation was probably associated with abnormal morphology and distribution of megakaryocytes and fibrosis.

[Features and clinical significance of gene mutations in patients with myelodysplastic syndromes with ring sideroblasts].

Objective: To explore the features and clinical significance of gene mutations in patients with myelodysplastic syndromes with ring sideroblasts (MDS-RS) . Methods: A total of 255 newly diagnosed primary MDS-RS patients were retrospectively reviewed from our center from January2001 to June 2019. SF3B1 gene mutations were detected by Sanger sequencing in 129 patients, and next generation sequencing (NGS) was performed in the other 126 patients using a set of selected 112-genes. Results: A total of 193 (75.7%) patients presented with SF3B1 mutation, predominantly mutant at amino acid position 700 (K700E) (n=147, 76.2%) . Non-SF3B1 gene mutations were TET2 (16.7%) , ASXL1 (14.3%) , U2AF1 (11.1%) , TP53 (7.9%) , SETBP1 (6.3%) , and RUNX1 (6.3%) . RS 5%-<15% patients had a higher SETBP1 mutation frequency than RS≥15% patients (21.4% vs 4.5%, P=0.044) . Mutation frequencies of other genes were similar in both groups (all P>0.05) . SF3B1 variant allele frequencies (VAF) had positive correlation with marrow RS percentage but without statistical significance in RS 5%-<15% group (P=0.078, r=0.486) . SF3B1 mutant patients presented with higher marrow RS percentage compared with wild-type patients[40.0% (15.0%-80.0%) vs 25.5% (15.0%-82.0%) , P<0.001], and SF3B1 VAF positively correlated with RS percentage (P=0.009, rs=0.261) in RS≥15% group. Age, ANC, PLT, mean RBC corpuscular volume, RS percentage, IPSS-R cytogenetics, and IPSS-R risk score were significantly different between patients with SF3B1 mutations and wild-type SF3B1 (all P<0.05) . Multivariable survival analyses adjusted by age and IPSS-R cytogenetics revealed that SF3B1 mutation was an independent favorable prognostic factor (HR=0.265, 95% CI 0.077-0.917, P=0.036) , and TP53 mutation was an adverse variable independent of SF3B1 mutation (HR=6.272, 95% CI 1.725-22.809, P=0.005) . According to the mutant status of SF3B1 and TP53, MDS-RS patients were categorized into 4 groups, namely, with SF3B1 and TP53 mutation, with wild-type SF3B1 and TP53, with wild-type SF3B1 but TP53 mutation, and with SF3B1 mutation but wild-type TP53. There was a significant difference for OS among these 4 groups (P<0.001) . The former 3 groups showed no significant difference in OS in multiple comparisons. However, the SF3B1 mutation but wild-type TP53 group had a better OS than wild-type SF3B1 but TP53 mutation group and wild-type SF3B1 and TP53 group, whereas a similar OS compared with SF3B1 and TP53 mutation group. Conclusion: SF3B1 mutations were prevalent in MDS-RS patients with the most common mutation at amino acid position 700 (K700E) . SF3B1 mutation was an independent favorable prognostic variable, whereas TP53 mutation was an independent adverse variable. SF3B1 mutation could coordinate with TP53 mutation for more sophisticated prognosis stratification in MDS-RS patients.

[Mean corpuscular volume ≤100 fl was an independent prognostic factor in patients with myelodysplastic syndrome and bone marrow blast<5 percent].

Objective: To explore the prognostic effects of mean corpuscular volume (MCV) in patients with myelodysplastic syndromes (MDS) . Methods: 321 newly diagnosed, untransfused primary MDS patients who administered from December 2009 to December 2017 were enrolled. The association of MCV with prognosis and several clinical features and genetic mutations were analyzed. Results: Patients were divided into MCV≤100 fl (n=148) and MCV>100 fl (n=173) cohorts. Median overall survival of patients with MCV≤100 fl was shorter than their counterparts (27 months vs 72 months, P<0.001) . In subgroup analysis, MCV≤100 fl patients had worse survivals in bone marrow blast <5% cohort (34 months vs not reached, P=0.002) , but not so in ≥5 % cohort (17 months vs 20 months, P=0.078) . MCV≤100 fl was still an independent adverse variable (HR=1.890, 95%CI 1.007-3.548, P=0.048) after adjusting for clinical and laboratory variables and mutation topography in bone marrow blasts<5% cohort. In bone marrow blasts<5% cohort, patients with MCV≤100 fl had higher hemoglobin levels [90 (42-153) g/L vs 78.5 (28-146) g/L, P=0.015].The proportions of Revised International Prognostic Scoring System (IPSS-R) high/very high risks and poor/very poor IPSS-R karyotypes were higher in MCV≤100 fl cohort (28.8% vs 10.8%, P=0.003; 24.7% vs 12.9%, P=0.049) . MCV≤100 fl cohort had more genetic mutations than those with MCV>100 fl though without significance (0.988 vs 0.769, P=0.064) . Mutated SF3B1 was less frequently in MCV≤100 fl cohort (4.7% vs 15.4%, P=0.018) . Conclusion: MCV≤100 fl was an independent adverse variable after adjusting for clinical and laboratory variables and mutation topography in MDS patients with bone marrow blasts<5%.

[Clinical implications and prognostic value of TP53 gene mutation and deletion in patients with myelodysplastic syndromes].

Objective: To explore the clinical implications and prognostic value of TP53 gene mutation and deletion in patients with myelodysplastic syndromes (MDS) . Methods: 112-gene targeted sequencing and interphase fluorescence in situ hybridization (FISH) were used to detect TP53 mutation and deletion in 584 patients with newly diagnosed primary MDS who were admitted from October 2009 to December 2017. The association of TP53 mutation and deletion with several clinical features and their prognostic significance were analyzed. Results: Alterations in TP53 were found in 42 (7.2%) cases. Of these, 31 (5.3%) cases showed TP53 mutation only, 8 (1.4%) cases in TP53 deletion only, 3 (0.5%) cases harboring both mutation and deletion. A total of 37 mutations were detected in 34 patients, most of them (94.6%) were located in the DNA binding domain (exon5-8) , the remaining 2 were located in exon 10 and splice site respectively. Patients with TP53 alterations harbored significantly more mutations than whom without alterations (z=-2.418, P=0.016) . The median age of patients with TP53 alterations was higher than their counterparts[60 (21-78) years old vs 52 (14-83) years old, z=-2.188, P=0.029]. TP53 alterations correlated with complex karyotype and International prognostic scoring system intermediate-2/high significantly (P<0.001) . Median overall survival of patients with TP53 alterations was shorter than the others[13 (95%CI 7.57-18.43) months vs not reached, χ(2)=12.342, P<0.001], while the significance was lost during complex karyotype adjusted analysis in multivariable model. Conclusion: TP53 mutation was more common than deletion in MDS patients. The majority of mutations were located in the DNA binding domain. TP53 alterations were strongly associated with complex karyotype and always coexisted with other gene mutations. TP53 alteration was no longer an independent prognostic factor when complex karyotype were occurred in MDS.

[MicroRNA-133b suppresses cell proliferation and invasion of esophageal squamous cell carcinoma via downregulating TAGLN2 expression].

Objective: To investigate the expression of microRNA-133b (miR-133b) in esophageal squamous cell carcinoma (ESCC), and explore its effect and the underlying molecular mechanisms on cell proliferation and invasion. Methods: Real-time quantitative PCR (qPCR) was used to examine miR-133b expression in 63 ESCC tissues and paired adjacent non-cancerous tissues, several ESCC cells (Eca109, EC9706, EC1, TE1, KYSE70) and normal esophageal epithelial cell Het-1A. MiR-133b mimic, inhibitor and negative control (NC) were transfected into TE1 cells. The effect of miR-133b on cell proliferation and invasion were determined by CCK-8 and Transwell assays, respectively. Subsequently, the target gene of miR-133b was predicted by online tools TargetScan and miRDB, which was verified by dual luciferase reporter assays. Finally, Western blot was utilized to detect the effects of miR-133b overexpression on expression of target gene TAGLN2 as well as EMT-related proteins E-cadherin, N-cadherin, Snail, Slug and Vimentin. Results: Relative levels of miR-133b in ESCC tissues (0.295±0.040) were significantly lower than those in adjacent non-cancerous tissues (1.002±0.011, P<0.001). The expression of miR-133b was tightly associated with clinical staging, lymph node metastasis and prognosis. Moreover, relative levels of miR-133b in ESCC cells Eca109, EC9706, EC1, TE1 and KYSE70 (0.679±0.031, 0.391±0.008, 0.236±0.016, 0.031±0.005 and 0.099±0.020) were evidently lower than that in normal esophageal epithelial cell Het-1A (1.005±0.016, all P<0.001). In TE1 cells, miR-133b mimic significantly increased the level of miR-133b to 6.199±0.627, and suppressed cell proliferation and invasion, whereas miR-133b inhibitor obviously decreased its expression to 0.182±0.023, and promoted cell proliferation and invasion. Most notably, the relative luciferase activities of miR-133b-mimic group (0.320±0.018) in TE1 cells transfected with TAGLN-3'UTR-WT were markedly lower than that in NC group (1.010±0.036, P<0.001), whereas those in TAGLN-3'UTR-MUT transfection cells were 1.019±0.056 and 1.008±0.021, respectively, showing no significantly statistical difference (P>0.05). Furthermore, miR-133b overexpression markedly downregulated TAGLN2, N-cadherin, Snail, Slug and Vimentin levels, and increased E-cadherin expression. Conclusion: MiR-133b plays an important role in the proliferation and invasion of ESCC cells by regulating TAGLN2 expression, and it may be a potential therapeutic target for ESCC patients.

[A study of clinical characteristics and prognosis of primary myelofibrosis patients with thrombocytopenia in varied degrees].

Objective: To evaluate clinical characteristics and prognosis of primary myelofibrosis (PMF) patients with thrombocytopenia in varied degrees. Methods: Clinical features and survival data of 1 305 Chinese patients with PMF were retrospectively analyzed. The prognostic value of thrombocytopenia in patients with PMF was evaluated. Results: 320 subjects (47%) presented severe thrombocytopenia (PLT<50×10(9)/L), 198 ones (15.2%) mild thrombocytopenia [PLT (50-99)×10(9)/L] and 787 ones (60.3%) without thrombocytopenia (PLT ≥ 100×10(9)/L). The more severe the thrombocytopenia, the higher the proportions of HGB<100 g/L, WBC<4×10(9)/L, circulating blasts ≥ 3%, abnormal karyotype and unfavourable cytogenetics (P<0.001, P<0.001, P=0.004, P<0.001 and P<0.001, respectively) were observed in this cohort of patients. The more severe the thrombocytopenia, the lower the proportion of JAK2V617F positive (P<0.001) was also noticed. Platelet count was positively correlated with splenomegaly, HGB and WBC (P<0.001, correlation coefficients were 0.131, 0.445 and 0.156, respectively). Platelet count was negative correlated with constitutional symptoms and circulating blasts (P=0.009, P=0.045, respectively; correlation coefficients were -0.096 and -0.056, respectively). The median survival of patients with severe thrombocytopenia, mild thrombocytopenia and without thrombocytopenia were 32, 67 and 89 months, respectively (P<0.001). Multivariate analysis identified thrombocytopenia in varied degrees (HR=1.693, 95%CI 1.320-2.173, P<0.001) and Dynamic Internation Prognostic Scoring System(DIPSS) prognostic model (HR=2.051, 95%CI 1.511-2.784, P<0.001) as independent risk factors for survival. Conclusion: PMF patients with severe thrombocytopenia frequently displayed anemia, leucopenia, circulating blasts and short survival, so active treatment measures should be taken especially in these patients.

[Mechanisms of the suppression of proliferation and invasion ability mediated by microRNA-147b in esophageal squamous cell carcinoma].

Objective: To explore the expression of microRNA(miR)-147b in esophageal squamous cell carcinoma (ESCC) and its regulatory roles in cell proliferation, cell cycle and invasion as well as its molecular mechanisms. Methods: Real-time quantitative PCR (qPCR) was used to investigate the expression of miR-147b in ESCC tissues and cells. Negative control (NC) and miR-147b inhibitor were transfected into ESCC EC1 and EC9706 cells, which were divided into two groups: NC group and miR-147b inhibitor group, and qPCR was employed to detect the miR-147 level and CCK-8. Flow cytometry and Transwell chamber were utilized to investigate the effects of miR-147b downregulation on cell proliferation, cell cycle and invasion in ESCC cells. Besides, target genes of miR-147b was confirmed by double luciferase reporter assay. Subsequently, qPCR and Western blot were used to examine the effects of miR-147b downregulation on NDUFA4 expression, and NDUFA4 expression and its correlation with miR-147b were investigated in ESCC tissues. Results: Relative level of miR-147b in ESCC tissues (3.03±0.27) and cells were markedly higher than that in para-carcinoma tissues (1.00±0.01) and normal esophageal epithelial cell, and the differences had statistical significance (P<0.01), and its high expression was closely associated with clinical staging, invasion depth, histological grading and lymph node metastasis(P<0.05). Importantly, clinical staging, lymph node metastasis and miR-147b may be an independent prognostic factor in ESCC. Moreover, miR-147b downregulation dramatically suppressed cell proliferation, arrested cell cycle in G0/G1 phase and reduced invasion ability in ESCC cells. Most importantly, NDUFA4 was a direct target gene of miR-147b, and miR-147b inhibitor evidently upregulated the expression of NDUFA4. Furthermore, NDUFA4 displayed low expression in ESCC tissues and its expression exhibited negative correlation with miR-147b expression. Conclusions: The downregulation of miR-147b expression significantly suppresses the proliferation and invasion abilities as well as alters cell cycle distribution in ESCC.

[Predictive and Prognostic significance of high-sensitivity modified Glasgow Prognostic Score (HS-mGPS) in advanced gastric cancer patients treated with neoadjuvant chemotherapy].

Objective: To study the predictive and prognostic significance of high-sensitivity modified Glasgow Prognostic Score (HS-mGPS) on the effect of neoadjuvant chemotherapy for advanced gastric cancer. Methods: 117 patients with advanced gastric cancer received neoadjuvant chemotherapy with SOX (oxaliplatin+ S1) or mFOLFOX 6(oxaliplatin+ CF+ 5-FU) regimen. HS-mGPS was calculated according to blood C-reactive protein (CRP) concentration and serum albumin (ALB) level. The correlation between HS-mGPS and clinicopathological characteristics was determined and the predictors of survival were analyzed. Results: 117 patients with stage ⅡB (43 cases), stage Ⅲ (60), and stage Ⅳ (14) received preoperative neoadjuvant chemotherapy. The overall response rate of neoadjuvant chemotherapy was 61.5%(72/117), and the tumor control rate was 88.0% (103/117), with a pathological response rate of 91.5% (107/117). The R0 resection rate was 81.2% (95/117). The median disease-free survival (DFS) was 21.0 (95% CI 6.4-35.6) months. The median overall survival (OS) was 39.0 (95% CI 21.4-56.6) months. Higher HS-mGPS was associated with higher T stage, local lymph-node metastasis, distant metastasis, lower chemotherapy overall response rate and lower pathological response rate (all P<0.05). The univariate analysis and multivariate analysis showed that higher HS-mGPS, presence of local lymph-node metastasis and non R0 resection were associated with poorer DFS and OS (P<0.05). Conclusion: HS-mGPS can be used to predict the benefits of neoadjuvant chemotherapy and as an independent prognostic factor for survival in patients with advanced gastric cancer.

[Gene mutations from 511 myelodysplastic syndromes patients performed by targeted gene sequencing].

Objective: To study the characteristics of gene mutations in Chinese myelodysplastic syndromes (MDS) patients. Methods: A total of 511 Chinese patients with MDS performed 112-gene targeted sequencing were retrospectively analyzed. Results: Eighty-three distinct mutant genes were found in 511 patients with MDS. Amongst these, the most frequent mutations was associated with epigenetics (50%) , followed by spliceosome (37%) , signal transduction (34%) , transcription factors (24%) and cell cycle/apoptosis (17%) . 439 subjects (86%) had at least one gene mutation. The mean number of mutations in refractory anemia with unilineage dysplasia (RCUD) was 1.25, refractory anemia with multilineage dysplasia (RCMD) was 1.73, refractory anemia with ring sideroblasts (RARS) was 2.79, refractory anemia with excess blasts-1 (RAEB-1) was 2.22, RAEB-2 was 2.34, MDS with isolated 5q- was 2.67, MDS, unclassified (MDS-U) was 2.00. U2AF1 mutant subjects were more likely to have isolated+8[Q<0.001, OR=4.42 (95% CI 2.23-8.68) ]and less likely to have complex karyotypes[Q=0.005, OR=0.22 (95% CI 0.04-0.72) ]. According to the number of gene mutations, all subjects were categorized into three groups, namely group with 0-1 mutation, with 2 mutations and with three or more mutations. There was a significant difference in overall survival (OS) among three groups (P=0.041) . Conclusion: About 90% patients with MDS have at least one gene mutation. Genes associated with epigenetics and spliceosome are most common mutated genes in MDS. The increased numbers of gene mutations accompany with disease evolution and associate with poor prognosis.

CARD9 mutation linked to Corynespora cassiicola infection in a Chinese patient.

Corynespora cassiicola is a plant pathogen associated with leaf-spotting disease. The fungus has been found on diverse substrates: leaves, stems and roots of plants; nematode cysts and human skin. It rarely causes human infections. Here we report one case of subcutaneous phaeohyphomycosis caused by C. cassiicola with prominent tissue necrosis in a woman. All of her clinical features pointed towards a genetic linkage. Hence, whole-exome sequencing and Sanger sequencing were performed on this patient. One mutation of CARD9 was detected.

Effect of Repeated Recruitment Manoeuvres on Patients with Severe Acute Respiratory Distress Syndrome.

OBJECTIVE: The study aimed to evaluate the influence of repeated recruitment manoeuvres (RRMs) on lung injury in patients with acute respiratory distress syndrome (ARDS). METHODS: Forty-one ventilated patients with severe ARDS were selected for this study. Recruitment manoeuvres (RMs) were conducted with continuous positive airway pressure (CPAP; 30 cm H2O for 40 seconds). Recruitment manoeuvres were repeated every two hours for all three groups. Changes in haemodynamics, pulmonary compliance, gas exchange and extravascular lung water index (EVLWI) were monitored before RM (pre-RM), 10 minutes after each RM, and four hours after RM3 (4 hours post-RRM). Pulmonary inflammatory factors (tumour necrosis factor-alpha [TNF-α] and interleukin [IL]-6 and -10) were also analysed. RESULTS: Compared with those in pre-RM, pulmonary compliance, oxygenation index (ratio of partial pressure of arterial oxygen to fraction of inspired oxygen [PaO2/FiO2]) and EVLWI remarkably improved in RM1, RM2, RM3 and 4 hours post-RRM (p < 0.05). The PaO2/FiO2 ratio increased significantly in RM1 and RM3 (p < 0.05). Extravascular lung water index decreased significantly in RM1 compared with that in RM3 and 4 hours post-RRM (p < 0.05). There was no significant difference in cytokines. CONCLUSION: Repeated recruitment manoeuvres during lung-protected ventilation can improve pulmonary compliance and oxygenation and significantly decrease extravascular lung water in ARDS patients. Lung injury was not worsened by RRMs in patients with severe ARDS.

Comparative study on 4 EIA kits for screening antibody to hepatitis C virus in pooled sera.

Four enzyme immunoassay (EIA) test kits, 1 Canadian product and 3 Chinese products, were used in the comparative study. Each pool consisted of 5 sera, and the 5 single sera were tested as controls. The tests were carried out according to the instructions, keeping the same dilution of each serum in single and pool samples. It was found that with the Canadian kit, the positive and negative results of pooled sera had no difference from that of the controls (P > 0.10). In the case of Chinese Yali and Kehua kits, the positive results of pooled sera showed no difference from the controls (P > 0.10), but the optical density (OD) of negative pools were increased (P < 0.01), though quite distant from the cut-off values. In the case of Changzheng kit, the OD of positive pools were significantly lower than those of the controls (P < 0.05), and weak positive samples missed the detection. However this problem could be overcome by blocking the microwells beforehand. Our experiment demonstrate that not all EIA test kits are suitable for screening pools for antibody to hepatitis C virus, and that it is important to assess the sensitivity of the EIA kit to be used for this purpose.

A prospective study of a serum-pooling strategy in screening blood donors for antibody to hepatitis C virus.

BACKGROUND: To examine the feasibility and to perform a cost-benefit analysis of a pooling protocol of enzyme immunoassay (EIA) screening for antibody to hepatitis C virus (anti-HCV) under real conditions, a prospective study was carried out using sera from 1875 local blood donors. STUDY DESIGN AND METHODS: In the absence of knowledge of the anti-HCV reactions, the donor's sera were pooled into groups of five consecutive samples for testing by EIA. The dilution and final volume of the serum pool were adjusted to equal those recommended for single-serum EIA by the manufacturer of the test kit. The results obtained were compared with those of single-serum EIA to assess the feasibility of the pooling protocol. By applying probability theory, the percentage of reduction in the number of tests performed (L value) when the serum-pooling strategy was used was calculated for several anti-HCV seroprevalences and for varied sizes of pool. The calculations were performed on a computer using a program compiled by the authors. RESULTS: The results showed that seroprevalence was 2.24 percent (95% CI, 1.57-2.91%); the rate of false negativity was 0 (95% CI, 0-8.4%), the sensitivity of the pooling protocol was 100 percent (95% CI, 91.6-100.0%), the rate of false positivity was 0.8 percent (95% CI, 0-1.8%), and the specificity of the pooling protocol was 99.2 percent (95% CI, 98.2-100.0%). Cost-benefit analysis showed that the pooling protocol could save 69.3 percent of the cost. A table of L values can be used conveniently by serologists to determine the optimum pool size if estimates of seroprevalence are available. CONCLUSION: The pool EIA did not perform worse than individual EIAs, and the pooling strategy was markedly less expansive. The pooling protocol was recommended for screening of anti-HCV-positive subjects from large populations with low seroprevalence.

Regulation of the human TRH (hTRH) gene by human thyroid hormone receptor beta 1 (hTR beta 1) mutants.

TRH is negatively regulated by T3 both in the hypothalamic paraventricular nucleus and transient transfection models. Mutations in hTR beta 1 genes are associated with the syndrome of generalized resistance to thyroid hormone. To investigate potential effects of mutant TRs on T3 regulation of the hTRH gene, transient gene expression assays were performed in human neuroblastoma (HTB-11) cells with an hTRH promoter-luciferase construct, wild type (WT) hTR beta 1, and three qualitatively distinct hTR beta 1 mutant forms (ED, OK and PV). In the presence of T3 (10(-9) M), liganded WT-hTR beta 1 inhibited hTRH promoter activity significantly (40%). Cotransfection of each of the two mutants (ED and OK) achieved similar levels of inhibition only at 10 to 100 fold increased T3 concentrations. Of interest, a 10x excess of mutant ED or OK could also exert dominant negative effects upon WT hTR beta 1-T3 mediated inhibitory actions on the hTRH promoter. In contrast, mutant TR-PV exerted neither inhibitory nor dominant negative effects at even higher concentrations of T3. Moreover, all three unliganded mutant forms stimulated TRH promoter activity significantly in the absence of T3, despite their different mutations in the ligand-binding domain (LBD). These data demonstrate that thyroid hormone resistance at the level of TRH gene regulation, due to reduced inhibitory actions of mutant TR-T3 complexes, as well as dominant negative effects upon WT hTR beta 1 mediated inhibition, likely contribute to elevated TSH values observed in the syndrome of thyroid hormone resistance.

Reversal of TR-T3 inhibition of the hTRH gene by excess TR ligand-binding domain: evidence for novel accessory protein.

The thyrotropin-releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T3). T3 positive regulatory effects on other target genes, such as the growth hormone gene, are mediated through heterodimerization of thyroid hormone receptors (TRs) with RXR or other auxiliary nuclear protein(s). To explore whether an accessory co-suppressor protein(s) may be involved in T3 inhibitory regulation of human TRH gene transcription, transient gene expression studies have been carried out using a hTRH-luciferase (TRH-Luc) chimetric reporter construct, an hTR beta 1 expression construct, and pABgal-hTR beta 1 ligand-binding domain (LBD) fusion constructs, cotransfected into a human neuroblastoma cell line (HTB-11,ATCC). Results herein indicate that T3-dependent inhibitory regulation (48-60% of control) of the hTRH gene promoter by hTR beta 1-T3 complexes could be abrogated completely by cotransfection of a 10 x excess of hTR beta 1-LBD (TR 168-456 aa) in a pABgal94 vector. In striking contrast, cotransfection of a 10 x excess of highly truncated hTR beta 1-LBD (TR 452-456 aa) failed to reverse T3-mediated TRH promoter inhibition. This squelching effect by excessive intact TR-LBD, moreover, could not be reversed by raising T3 concentration 100-fold (from 10(-8) to 10(-6) M), thus excluding a squelching effect of T3 itself by excess LBD. These results suggest that negative regulation of the hTRH gene promoter activity by TR beta 1-T3 complexes involves interactions with an accessory co-suppressor protein, which may bridge DNA-bound TR beta 1-T3 complexes to the transcriptional initiation complex.

Hippocampal input to the hypothalamus inhibits thyrotrophin and thyrotrophin-releasing hormone gene expression.

We have used in situ hybridization histochemistry to determine the effect of hippocampal-hypothalamic disconnection on hypothalamic thyrotrophin-releasing hormone (TRH) and anterior pituitary thyrotrophin beta subunit (TSH beta) transcripts in adult male CFY rats. Electrothermal lesions of the fornix pathway significantly increased TRH and TSH transcripts and increased circulating levels of triiodothyronine (T3). Fornix transection did not, however, prevent feedback regulation of TRH and TSH transcripts during exogenous T3-induced hyperthyroidism or propylthiouracil-induced hypothyroidism. Hippocampal inputs to the hypothalamus contribute to setting the basal activity of the thyroid axis, but do not mediate the feedback effects of T3.

The effect of dietary protein on thyrotropin-releasing hormone and thyrotropin gene expression.

In the rat, 48 h of food deprivation significantly reduces hypothalamic paraventricular nucleus (PVN) thyrotropin-releasing hormone (TRH) gene expression, anterior pituitary thyrotropin (TSH) gene expression and circulating triiodothyronine (T3). Using in situ hybridization histochemistry, we have now assessed the effect of selective nutritional deprivation, by comparing protein-free and protein and fat-free diets with a normal diet matched for total energy content. As previously demonstrated, fasting markedly reduced PVN TRH transcripts, pituitary TSB beta transcripts, circulating T3 and body weight. Compared to rats fed a control diet, rats fed a protein-free or a protein and fat-free diet of similar energy content showed a highly significant decrease in PVN TRH transcripts, pituitary TSB beta transcripts and circulating T3 levels. The exclusion of fat from the protein-free diet did not produce any further decline in the parameters measured. This indicates that variations in the protein composition alone of the diet are sufficient to reduce hypothalamic TRH mRNA, pituitary TSB beta mRNA and plasma T3, and are the predominant factors in the TRH response to starvation.

[Primary gastric lymphoma: a clinical and pathologic analysis of 25 cases].

25 cases of primary gastric lymphoma diagnosed in our hospital from 1973 to 1987 were analysed. Primary gastric lymphoma comprised 1.55% of the stomach malignancies. There were 25 gastric lymphomas among 198 patients with lymphomas (12.6%). The diagnostic accuracy with X-ray was 30.4%. However, with modern fiberoptic endoscopy and directed biopsy technique, a correct diagnosis was made in 72% of the cases. Of the 24 follow-up cases, the overall 5 years survival rate was 54.28%. Surgery combined with radiotherapy or chemotherapy has yield a 5-year survival rate of 72%. Therefore, both correct and early diagnosis may contribute to improved prognosis.