Differential gene expression in genetically matched mouse melanoma cells with different metastatic potential.

In vitro fusion of weakly metastatic Cloudman S91 melanoma cells with macrophages from DBA/2J mice (syngeneic with Cloudman S91 melanoma) produced hybrids with metastatic potentials ranging from low to high, with more than half showing enhanced metastasis over the parental melanoma [Clin. Exp. Metastasis 16 (1998) 299]. These hybrids, derived from the same parental fusion partners, represent a unique genetically matched model for analyzing differential gene expression regulating the metastatic phenotype. We have examined the differences in gene expression in metastatic fusion hybrid compared to its parental partners, non-/poorly metastatic melanoma cells and normal macrophages. An approach by selective polymerase chain reaction (PCR) amplification and display of 3' end restriction fragments of double-stranded cDNAs was used [Methods Enzymol. 303 (1999) 272]. Gene expression analyses showed an extensive set of transcripts that were up- or down-regulated in the most metastatic hybrid, H95-1, compared to the parental macrophages or melanoma cells. Sequence analyses of more than 60 of these differentially expressed cDNAs revealed significant up- or down-regulation of a number of genes known to be associated with metastasis of melanoma and other solid tumors. Some genes are found to express exclusively either in normal macrophages or in melanoma. Thirteen fragment sequences were found with no matches with GenBank search. Comparison of these gene expression patterns should be of great value in understanding the coordinate programs regulating metastasis. Further, the increased expression of gene(s) common in macrophage and fusion hybrids may be of importance in identifying the regulatory factor(s) related to macrophage-like trait, motility, a critical step of metastatic processes, in hybrids.

Differential display of protein tyrosine kinase cDNAs from human fetal and adult brains.

Here we describe a differential display method for surveying the expression of most protein tyrosine kinases and applying it to cDNAs from human fetal and adult brains. The method involves two selective steps for processing the mRNA. At each step, degenerate oligonucleotide primers derived from highly conserved regions of the catalytic domain of the kinases are used. In the display with BstYI and BsiHKI digests of the cDNA, 65% and 59% of a total of 72 and 63 bands, respectively, represented fragments from a total of 27 different tyrosine kinases. The expression levels of the kinases in the display were comparable with those measured by RT-PCR. This method offers a relatively specific way to display differentially expressed gene families in any tissue and cell type.

Genomic and proteomic analysis of the myeloid differentiation program.

Although the mature neutrophil is one of the better characterized mammalian cell types, the mechanisms of myeloid differentiation are incompletely understood at the molecular level. A mouse promyelocytic cell line (MPRO), derived from murine bone marrow cells and arrested developmentally by a dominant-negative retinoic acid receptor, morphologically differentiates to mature neutrophils in the presence of 10 microM retinoic acid. An extensive catalog was prepared of the gene expression changes that occur during morphologic maturation. To do this, 3'-end differential display, oligonucleotide chip array hybridization, and 2-dimensional protein electrophoresis were used. A large number of genes whose mRNA levels are modulated during differentiation of MPRO cells were identified. The results suggest the involvement of several transcription regulatory factors not previously implicated in this process, but they also emphasize the importance of events other than the production of new transcription factors. Furthermore, gene expression patterns were compared at the level of mRNA and protein, and the correlation between 2 parameters was studied. (Blood. 2001;98:513-524)

RNA expression patterns change dramatically in human neutrophils exposed to bacteria.

A comprehensive study of changes in messenger RNA (mRNA) levels in human neutrophils following exposure to bacteria is described. Within 2 hours there are dramatic changes in the levels of several hundred mRNAs including those for a variety of cytokines, receptors, apoptosis-regulating products, and membrane trafficking regulators. In addition, there are a large number of up-regulated mRNAs that appear to represent a common core of activation response genes that have been identified as early-response products to a variety of stimuli in a number of other cell types. The activation response of neutrophils to nonpathogenic bacteria is greatly altered by exposure to Yersinia pestis, which may be a major factor contributing to the virulence and rapid progression of plague. Several gene clusters were created based on the patterns of gene induction caused by different bacteria. These clusters were consistent with those found by a principal components analysis. A number of the changes could be interpreted in terms of neutrophil physiology and the known functions of the genes. These findings indicate that active regulation of gene expression plays a major role in the neutrophil contribution to the cellular inflammatory response. Interruption of these changes by pathogens, such as Y pestis, could be responsible, at least in part, for the failure to contain infections by highly virulent organisms.

[The clinical feature of febrile episodes].

Institutionalized elderly patients are at risk of nosocomial infection because of their compromised status by aging. To clarify the relationship between fever and disease in elderly patients, we analyzed 1,105 febrile episodes, the etiology of which were already diagnosed, of 443 patients (136 men, 307 women). All patients who were 65 years of age or older and who had been admitted to the hospital for more than 7 days had fevers above 37.5 degrees C recorded. The etiologies of the 1,105 febrille episodes were respiratory tract infection in 381 (34.5%), urinary tract infection in 263 (23.8%), other diseases in 164 (14.8%) and in 297 (26.9%) unknown. The episodes were categorized into two groups by the degree of initial fever: group A, 559 episodes (50.6%) of 37.5-38.0 degrees C and group B, 546 episodes (49.4%) of above 38.0 degrees C. Of the episodes, 41.0% were one-day fevers, 21.4% two-day fevers, and 14.0% three-day fevers. The frequency of two-or-more-day fevers was significantly higher in group B (69.0%) than in group A (49.2%) (p < 0.001). In group B, respiratory tract infection (44.3%) was more frequent than urinary tract infection (16.1%) (p < 0.001). Of the respiratory tract infections, 63.5% were in group B and, in contrast, 66.5% of the urinary tract infections were in group A. The white blood cell count and C-reacting protein levels were significantly higher in group B than in group A (p < 0.001). The degree of initial fever is an important predictive marker of severity of disease in elderly patients.

[Antibody response to a single injection of influenza vaccine among geriatric inpatients vaccinated annually].

To determine the efficacy of a single influenza vaccine administration in the elderly receiving annual influenza vaccination, antibody response to influenza vaccine was compared between once and twice injections in a geriatric cohort. Influenza vaccination had been done for 69 inpatients in the year prior to the study, and was administered twice for 34 of them and once for the other 35 during the study period. Influenza vaccine was injected twice to 77 inpatients who had not received influenza vaccine in the year prior to the study. Hemoagglutination inhibition (HI) antibody titer for influenza A/H1N1, A/H3N2, and B was measured before vaccination, after the first vaccination, after the second vaccination, and after the epidemic period, September 1995 to April 1996. HI antibody titer prior to vaccination was significantly higher in the patients who had received influenza vaccination the previous year. The influenza vaccine induced an increase in HI titer in almost all subjects, and the geometric mean of the HI titer after vaccination in the patients who received vaccine once was comparable to that of the patients injected vaccine twice. The number of patients with HI titers of over 128x increased, and the frequency ranged from 60.0% to 97.1% for the influenza viruses of the three subtypes. The frequency of HI titers over 128x was not significantly different among the three groups. The second vaccination did not increase the number of patients with HI titers over 128x when compared with the number after the first injection in the patients who had received influenza vaccine the previous year. These results suggest that prior vaccination does not diminish the antibody response to influenza vaccine in the elderly. The efficacy of a single influenza vaccination is comparable to that achieved by twice injections in the elderly receiving annual influenza vaccination.

[Febrile episodes in elderly inpatients--a one year survey to determine the causes of fever in hospital].

To determine the cause of nosocomial infections, all febrile episodes of hospitalized elderly patients aged 65 and older at a hospital in Fukuoka City were categorized between April 15 1994 and April 14 1995. A febrile episode was a temperature above 37.5 degrees C after 7 consecutive days of normal body temperature (below 37.5 degrees C). Various clinical tests including blood examination, urinalysis, chest radiography and bacterial culture were done on the first and 7th day of the fever. A total of 1105 episodes in 443 patients (male 136, female 307) fulfilled this criteria for fever. The fevers were mainly due to respiratory tract (381 cases, 34.5%) and urinary tract infections (263 cases, 23.8%). There were 135 infections (12.2%) of other kinds and 297 cases (26.9%) that were classified as unknown. Approximately 70% of the febrile episodes were caused nosocomial infections, suggesting increased risk of infection in the hospitalized elderly and the importance of early detection of febrile changes in elderly inpatients.

[Impact of influenza epidemics and efficacy of vaccination among geriatric inpatients].

To determine the impact of influenza epidemics among geriatric inpatients and to monitor the clinical efficacy of influenza vaccination, the influenza infection rate in non-vaccinated inpatients was determined serologically and the incidence of febrile episodes and death were compared between the vaccinees and non-vaccinees hospitalized in the referred hospital from January through September, 1995. Three influenza subtypes, influenza A/H1N1, A/H3N2, and B, were endemic simultaneously from January to March in 1995. The pattern of incidence of febrile episodes varied for each ward. A total of 123 non-vaccinated inpatients were tested for elevation of serum hemagglutination inhibition titer to the three subtypes of influenza virus. Of these, 58 (47.2%) patients were infected with at least one of the influenza viruses during the epidemic of 1995. No patient with pre-existing HI titer over 128X was infected with any of three types of influenza, indicating that HI titer over 128X is the protective level. The febrile episode frequency was significantly higher in the non-vaccinees than in the vaccinees (49.6% vs. 32.6%), but it was quite comparable in the two groups after the influenza epidemic (34.9% vs. 35.8%). The number of observed deaths from January to September of 1995 was 4 (4.9%) in the vaccinee group and 12 (9.8%) in the non-vaccinee group. These results suggest that influenza epidemics have a striking impact on geriatric inpatients and that influenza vaccination has significant efficacy for the reduction of harmful events associated with influenza infection.

[Effect of the prior influenza vaccination on serum antibody titer induction by subsequent inactivated influenza vaccine in the elderly].

In order to investigate the effects of prior influenza vaccination on subaequent annual influenza vaccination in the geriatric population, we analyzed serum hemagglutinine inhibition antibody tirers (HI titer) before and after vaccination with inactivated influenza vaccine in elderly inpatients. A total of 163 inpatients of 60 years or older were enrolled with informed consent. They were classified by vaccination status in the previous year, 53 patients had inactivated vaccine (inactivated). 52 patients had genetically assorted cold-adapted influenza live attenuated vaccine (cold-adapted), and 53 had no influenza vaccine history during the past year. The HI titer was higher in the inactivated group than in the cold-adapted and non-vaccinated groups, suggesting residual immunological effects of inactivated influenza vaccine from the previous year vaccination. The HI titer after the inactivated vaccine in 1993 was higher in both the inactivated and cold-adapted groups than in the non-vaccinated group. The number of patients with HI titers of 2(7) or higher, which is the putative protective HI titer level for influenza infection, was significantly higher in both the inactivated and cold-adapted groups than the non-vaccinated group. These results suggest that continuous annual influenza vaccination does not impair the effects of vaccination, and may actively promote elevated HI titers.

[A case of an elderly patient with dementia and gait disturbance associated with influenza].

We report a case of progressive dementia and prolonged gait disturbance correlated with influenza A/H3N2 infection in 91-year-old female patient, admitted because of in ability to take care of herself due to aging and cerebral infarction. At admission, conversation and comprehension were not significantly impaired, and she was able to walk by herself. Flu symptoms such as high grade fever, chills, arthralgia, and cough appeared after a short stay at home. Influenza A/ H3N2 was confirmed serologically. Delirium occurred on the sixth day after influenza onset, persisted for three weeks, followed by recovery. Dementia symptoms such as memory defects and disorientation continued and did not improve. Due to this febrile episode, she was unable to walk unassisted. The results of computed tomography performed before and after the influenza episode were unremarkable for additional cellebro-vascular events during the observed period. Influenza infection may be an important risk factor for reducing the quality of life in the elderly. In geriatric cases, influenza should not be treated as a mere transient illness, but rather one which has important consequences for the elderly population, including the possibility of life threatening complications.

[Clinical significance of peak body temperature, white blood cell count, and C-reactive protein level in febrile episodes among geriatric inpatients].

To investigate the clinical implication of peak body temperature, peripheral blood white blood cell (WBC) count, and serum C-reactive protein (CRP) level in febrile symptoms among geriatric hospitalized patients, they were analyzed in 968 febrile episodes obtained from 433 hospitalized patients in the referred hospital. Episodes of one day duration were most frequent (41.6%). WBC count was elevated over 8000/microliters in 475 episodes (49.1%) and CRP exceeded 1.0 mg/dl in 770 episodes (79.5%). Frequency of WBC elevation decreased and frequency of CRP elevation increased according to the time course. The mean value of CRP increased significantly according to the time course. The frequency of WBC count increase and CRP elevation and their averages correlated to the peak body temperature. The peak body temperature displayed the most striking correlation to the length of febrile episodes among three clinical indicators, peak body temperature, WBC count, and CRP level. These results indicate that the elevation of WBC count and/or CRP level is frequent in geriatric patients with febrile symptoms. Peak body temperature may serve as a clinical indicator of the severy of the febrile disease occurring in geriatric patients.

[Serum albumin level as a predictor of incidence of febrile episodes and mortality in hospitalized geriatric patients].

To investigate the relationship between serum albumin level and incidence of febrile episodes and mortality in the elderly, we studied 748 patients hospitalized for over one year. The subjects included 123 males and 355 females with a mean age 81.2 years. The average serum albumin level was 3.79 g/dl and levels of serum albumin decreased with advancing age. The incidence of febrile episodes was 1.8 per year in patients with serum albumin levels over 4.1 g/dl, increasing with decline of serum albumin levels. The incidence of febrile episodes was 5.3 per year in patients with serum albumin levels under 3.0 g/dl. Patients with serum albumin levels under 3.0 g/dl displayed a high incidence of febrile episodes irrespective of age. Age adjusted in-hospital mortality was 40.4% during the observed period in patients with serum albumin levels under 3.0 g/dl, significantly higher than that of the patients with serum albumin levels over 3.1 g/dl. Relative risk of febrile episode and mortality calculated using the patients with serum albumin levels over 4.1 g/dl as a control was 2.9 and 2.1, respectively, in the patients with serum albumin levels under 3.0 g/dl. These results indicate that serum albumin level is a simple, but strong, predictor of susceptibility of febrile episode and death. Patients with serum albumin levels under 3.0 g/dl may constitute a high risk group for febrile episode and death.

[Incidence and duration of febrile episodes in a hospitalized geriatric cohort].

Fever is a common and important clinical symptom observed among hospitalized geriatric patients. To investigate the frequency and duration of fever episodes, we surveyed fever episodes in a hospital where the frequency of patients over 60 years of age exceeds 90 per cent of the patients. Fever episodes with body temperature of over 37.5 degrees C were registered from May in 1991 to December in 1994, and 6809 episodes were subjected to analysis. The average incidences per month were 157.1, 165.3, 158.0, and 139.3 in 1991, 1992, 1993, and 1994, respectively. The numbers of episodes per month did not show any significant correlation with temperature or humidity. Average duration of the episodes were 8.0, 6.5, 7.6, and 6.7 days for 1991, 1992, 1993, and 1994, respectively. Episodes of one day duration were the most frequent in all months, and the frequencies of that ranged from 37.1% to 58.6% with a mean of 47.8%. The average duration of episodes and the frequency of one day episodes did not change significantly irrespective of a notable decrease in the total incidence. The high frequency of one day episodes and their consistency through the observed period suggest that fevers with one day duration are one of the characteristic features of the febrile symptoms in geriatric patients. Causality and prevention methods for these one day fever episodes should be investigated.

[An outbreak of influenza A (H3N2) among hospitalized geriatric patients].

An outbreak of influenza A (H3N2) in a hospital where almost 90% of the inpatients are aged over 70 years is described. An increase of febrile episodes was seen during the period from January 29th to March 17th, 1992 in two of six wards paired sera, at the onset of fever and more than two weeks later, were obtained from patients in sixty-five episodes. Serum antibody titer to influenza A (H3N2) elevated over four times in 39 (60%) of 65. Influenza A (H3N2) virus was also isolated from seven patients. These results indicated an outbreak of influenza A (H3N2) in this population. Maximum body temperature was over 39 degrees C in 18 patients (46.2%) with influenza, and were significantly higher than that of the non-influenza patients. The duration of fever in 12 patients of 39 was longer than 8 days in 12 patients, and significantly longer than that of non-influenza cases. Bronchopneumonia was found in ten patients (25.6%). These results suggest that the influenza infection causes a high grade fever in geriatric patients and lasts longer than is commonly seen in patients with fever not associated with influenza. Influenza infection also frequently induces bronchopneumonia and may contribute to increase mortality in the elderly, especially in patients over 70 years old.

[A case of toxoplasmosis with dermatomyositis].

We report a case of dermatomyositis (DM) in a 15-year-old female with toxoplasmosis after ingestion of raw bovine liver. Facial erythema and cervical lymphadenopathy preceded myalgia and muscle weakness of the extremities. The diagnostic criteria of DM was fulfilled because of symmetrical and proximal dominant muscle weakness, elevation of myogenic enzyme (CPK, GOT, LDH, myoglobin, aldorase), myogenic pattern of electromyogram, skeletal muscle biopsy showing interstitial myositis with mild destruction of muscle fiber, and facial erythema. Immunological findings showed IgG anti-toxoplasma antibody to be 1340 IU/ml and IgM to be 7.0 (Cut off index 0.7), suggesting acute toxoplasmosis. Treatment with prednisolone for DM and acetylspiramycin for toxoplasmosis was successful. Toxoplasmosis should be considered as a possibility in patients with myositis.

Endothelin receptor in microvessels isolated from human meningiomas: quantification with radioluminography.

1. We characterized specific 125I-endothelin-1 (125I-ET-1) binding sites in microvessels isolated from human meningiomas, using an in vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system. 2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 +/- 0.3 nM; maximum binding capacity, 185 +/- 56 fmol/mg; means +/- SE for nine tumors). 3. In five cases of meningiomas, ET-3 competed for 125I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC50) of 1.6 +/- 0.4 x 10(-6) M], and a selective ETB receptor agonist, sarafotoxin S6c, up to 10(-6) M, did not displace ET binding from the sections. 4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC50 of 1.1 +/- 0.2 x 10(-9) M for the high-affinity component and 1.6 +/- 0.3 x 10(-6) M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC50 = 2.3 +/- 0.2 x 10(-10) M) and 10(-6) M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3. 5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ETA (non-ETB) receptors with a small proportion of ETB receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.

Enhanced expression of an endothelin ETA receptor in capillaries from human glioblastoma: a quantitative receptor autoradiographic analysis using a radioluminographic imaging plate system.

We identified and characterized 125I-endothelin-1 (125I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (KD = 4.7 +/- 1.0 x 10(-10) and 1.6 +/- 0.3 x 10(-10) M, respectively; Bmax = 161 +/- 38 and 140 +/- 37 fmol/mg, respectively; mean +/- SEM, n = 5). BQ-123, a selective antagonist for the ETA receptor, potently competed for 125I-ET-1 binding to sections of the microvessels with IC50 values of 5.1 +/- 0.3 and 5.1 +/- 1.5 nM, and 10(-6) M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC50 = 5.7 +/- 2.5 x 10(-10) and 1.4 +/- 0.2 x 10(-6) M for tumor microvessels, 1.8 +/- 0.6 x 10(-10) and 1.1 +/- 0.3 x 10(-6) M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ETA subtype.

A selective endothelin ETA antagonist, BQ-123, inhibits 125I-endothelin-1 (125I-ET-1) binding to human meningiomas and antagonizes ET-1-induced proliferation of meningioma cells.

1. We studied the effects of BQ-123, a selective ETA receptor antagonist, on 125I-endothelin-1 (125I-ET-1) binding to cell surface receptors in surgically exercised human meningiomas and on ET-1-induced DNA synthesis in cultured human meningioma cells in vitro, using a quantitative receptor autoradiographic technique with radioluminography and 3H-thymidine incorporation, respectively. 2. All of the human meningiomas expressed high-affinity binding sites for 125I-ET-1, regardless of differences in histological subtypes (Kd = 2.6 +/- 0.2 nM, Bmax = 374 +/- 93 fmol/mg; mean +/- SE; n = 9). 3. BQ-123 competed for 125I-ET-1 binding to sections of meningiomas with IC50S of 3.2 +/- 0.9 x 10(-7) M, and 10(-4) M BQ-123 displaced 80% of the binding. 4. ET-1 significantly stimulated DNA synthesis in cultured human meningioma cells, up to 170% of the basal level in the presence of 10(-9) M ET-1. BQ-123 inhibited ET-1 (10(-9) M)-induced DNA synthesis in meningioma cells, in a dose-dependent manner, and 10(-5) M BQ-123 reduced it to 120% of the basal level. 5. The number of meningioma cells determined after 4 days in culture was dose dependently increased in the presence of ET-1 (10(-9) and 10(-7) M). The growth rate of meningioma cells, incubated with 10(-9) M ET-1, was reduced by 50% in the presence of 10(-7) M BQ-123.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic effects of suramin on 125I-epidermal growth factor binding to human meningiomas.

1. We studied the effects of suramin, a nonspecific growth factor antagonist, on epidermal growth factor (EGF) binding to cell surface receptors in surgically excised human meningiomas, using quantitative receptor autoradiographic methods with radioluminography. 2. High concentrations (10(-4) - 10(-2) M) of suramin inhibited 125I-EGF binding to meningioma sections with IC50's of 3.2 +/- 0.4 x 10(-4) M, whereas lower concentrations (10(-5) - 10(-4) M) of the drug significantly enhanced EGF binding to the tumor. Scatchard analysis of EGF binding profile revealed significant increases in binding affinity following incubation in the presence of 5 x 10(-5) M suramin, without significant alterations in maximal binding capacity. 3. The addition of 10(-3) M suramin to the incubation buffer rapidly dissociated 125I-EGF previously bound to meningioma tissues as a function of time (dissociation half-life, T1/2 = 12.4 min). 4. Preincubation in the presence of 5 x 10(-5) M suramin resulted in significant increases in the subsequent binding of 125I-EGF to meningiomas, compared to findings in the control. 5. Our data indicate that (a) suramin exerts biphasic effects on EGF binding to the tissue sections of meningiomas in vitro, depending on the concentration of the drug; and (b) low concentrations of suramin enhance the affinity of the EGF receptor in the tumor sections, probably by interacting with the EGF receptor molecule rather than with the EGF peptide. 6. The functional role of increased EGF receptor affinity in meningioma sections in the presence of lower concentrations of suramin remains to be determined.