CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.     

Defective expression of the DNA mismatch repair protein, MLH1, alters G2-M cell cycle checkpoint arrest following ionizing radiation

A role for the Mut L homologue-1 (MLH1) protein, a necessary component of DNA mismatch repair (MMR), in G2-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G1 arrest and/or G1-S transition after damage was, however, not discounted. We report that MLH1-deficient human colon carcinoma (HCT116) cells showed decreased survival and a concomitant deficiency in G2-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected human colon carcinoma (HCT116 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embryonic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from MLH1 knockout mice also demonstrated classic DNA damage tolerance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLH1+) compared with HCT116 (MLH1-) cells after IR or 6-TG. Retroviral vector-mediated expression of the E6 protein did not, however, affect the enhanced G2-M cell cycle arrest observed in HCT116 3-6 compared with MLH1-deficient HCT116 cells. A role for MLH1 in G2-M cell cycle checkpoint control, without alteration in G1, after IR was also suggested by similar S-phase progression between irradiated MLH1-deficient and MLH1-proficient human or murine cells. Introduction of a nocodazole-induced G2-M block, which corrected the MLH1-mediated G2-M arrest deficiency in HCT116 cells, clearly demonstrated that HCT116 and HCT116 3-6 cells did not differ in G1 arrest or G1-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G1 cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLH1-mediated G2-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells.     

Interdisciplinary treatment for fibromyalgia syndrome: clinical and statistical significance

ATM has been identified as a gene that is responsible for ataxia telangiectasia (AT), a pleiotropic disorder of autosomal recessive inheritance. While many mutations of this gene in AT patients of various ethnicities have been reported, data on Japanese patients are scarce. In this report, we present the results of a thorough survey of ATM mutations in 14 unrelated AT patients, with an emphasis on Japanese subjects. We used a hierarchical strategy in which we extensively analyzed the entire coding region of the cDNA. In the first stage, point mutations were sought by PCR-SSCP in short patches. In the second and third stages, the products of medium- and long-patch PCR, each covering the entire region, were examined by agarose gel electrophoresis to search for length changes. We found a total of 15 mutations (including 12 new) and 4 polymorphisms. Abnormal splicing of ATM was frequent among Japanese, and no hotspot was obvious, suggesting no strong founder effects in this ethnic group. Eleven patients carried either one homozygous or two compound heterozygous mutations, one patient carried only one detectable heterozygous mutation, and no mutation was found in two patients. Overall, mutations were found in at least 75% of the different ATM alleles examined. Possible reasons for the inability to detect mutations in some patients are discussed.     

External causes of death among persons with developmental disability: the effect of residential placement

The authors analyzed death rates from external causes (accidents, injuries, homicides, etc.) for persons with developmental disability in California. There were 520 such deaths during the 1981-1995 study period, based on 733,705 person-years of exposure; this represents all persons who received any services from the state. Compared with the general California population, persons with developmental disability were at lower risk of homicide, suicide, and poisonings (standardized mortality ratios, 0.31-0.68), but higher risk of pedestrian accidents, falls, fires, and, especially, drowning (standardized mortality ratio=6.22). A major focus of the study was comparisons between different residential settings. Persons in semi-independent living had significantly higher risk than did those in their family home or group homes, with homicides rates being three times higher and pedestrian accidents rates being doubled, while persons in institutions had much lower risks with respect to most causes. Of the 28 deaths due to drug and medication overdoses, 79 percent occurred in supported living or small-group homes. Avoidable deaths could be reduced by making direct care staff more aware of the risks and better trained in acute care, along with improved monitoring of special incidents.     

Cytokine regulation by virus infection: bovine viral diarrhea virus, a flavivirus, downregulates production of tumor necrosis factor alpha in macrophages in vitro

Bovine bone marrow-derived macrophages were infected in vitro with noncytopathic or cytopathic strains of bovine viral diarrhea virus. Infection with both biotypes resulted in a decreased production of tumor necrosis factor alpha upon stimulation with heat-inactivated Salmonella dublin or lipopolysaccharide. Other macrophage functions were not downregulated, indicating that the observed effect was not due to a loss in macrophage viability. The downregulated production of tumor necrosis factor alpha in infected macrophages may contribute to the well-documented immunosuppression in animals infected with bovine viral diarrhea virus.     

Epidemiology of osteosarcoma and Ewing's sarcoma in childhood: a study of 305 cases by the Children's Cancer Group

BACKGROUND: The Children's Cancer Group conducted a case-control study to determine the role of a broad range of environmental and familial factors in the etiology of Ewing's sarcoma and osteosarcoma in children. These factors included radiation exposure and, for children with osteosarcoma, parental exposure to beryllium. METHODS: The parents of 152 children with osteosarcoma and 153 children with Ewing's sarcoma were interviewed by telephone. Controls were obtained by random digit dialing and were matched to cases by age and race. RESULTS: Female osteosarcoma patients had earlier onset of breast development (age 11.4 vs. 11.8 years, P=0.03) and menarche (age 12.1 vs. 12.5 years, P=0.002) but no significant differences in growth, whereas male osteosarcoma patients were similar in age at the onset of secondary sexual characteristics but reported significantly less weight gain during their growth spurt (6.6 vs. 11.7 kg, P=0.003). For children with Ewing's sarcoma, the growth spurt began earlier (age 12.1 vs. 12.7 years, P=0.12) and resulted in less weight and height gain (5.2 vs. 9.7 kg, P=0.002, and 10.2 vs. 12.7 cm, P=0.02, respectively) for males, but no differences were observed among females. For factors not related to growth and development (including a wide range of occupational, medical, and household exposures), there was little evidence of an etiologic role with respect to either tumor type. CONCLUSIONS: Differences between cases and controls with respect to growth and development showed no consistent pattern. This study did not identify any important risk factors for either type of childhood bone tumor.     

Decreased epinephrine responses to hypoglycemia during sleep

BACKGROUND: In patients with type I diabetes mellitus, hypoglycemia occurs commonly during sleep and is frequently asymptomatic. This raises the question of whether sleep is associated with reduced counterregulatory-hormone responses to hypoglycemia. METHODS: We studied the counterregulatory-hormone responses to insulin-induced hypoglycemia in eight adolescent patients with type I diabetes and six age-matched normal subjects when they were awake during the day, asleep at night, and awake at night. In each study, the plasma glucose concentration was stabilized for 60 minutes at approximately 100 mg per deciliter (5.6 mmol per liter) and then reduced to 50 mg per deciliter (2.8 mmol per liter) and maintained at that concentration for 40 minutes. Plasma free insulin, epinephrine, norepinephrine, cortisol, and growth hormone were measured frequently during each study. Sleep was monitored by polysomnography. RESULTS: The plasma glucose and free insulin concentrations were similar in both groups during all studies. During the studies when the subjects were asleep, no one was awakened during the hypoglycemic phase, but during the final 30 minutes of the studies when the subjects were awake both the patients with diabetes and the normal subjects had symptoms of hypoglycemia. In the patients with diabetes, plasma epinephrine responses to hypoglycemia were blunted when they were asleep (mean [+/-SE] peak plasma epinephrine concentration, 70+/-14 pg per milliliter [382+/-76 pmol per liter]; P=0.3 for the comparison with base line), as compared with when they were awake during the day or night (238+/-39 pg per milliliter [1299+/-213 pmol per liter] P=0.004 for the comparison with base line, and 296+/-60 pg per milliliter [1616+/-327 pmol per liter], P=0.004, respectively). The patients' plasma norepinephrine responses were also reduced during sleep, whereas their plasma cortisol concentrations did not increase and their plasma growth hormone concentrations increased slightly. The patterns of counterregulatory-hormone responses in the normal subjects were similar. CONCLUSIONS: Sleep impairs counterregulatory-hormone responses to hypoglycemia in patients with diabetes and normal subjects.     

Strain gauge plethysmography and Doppler ultrasound in the measurement of limb blood flow

The maintenance of adequate oxygen delivery (DO2) and tissue uptake (VO2) has become central dogma in the management of the critically ill. However, these parameters are derived using gas tensions measured in mixed venous blood and may not reflect changes in regional blood flow. Therefore, it has become necessary to provide estimates of blood flow to specific organs and to evaluate the most adequate techniques available. In order to define the best means of assessing blood flow to the lower limb noninvasively in normal subjects, measurements of superficial femoral arterial blood flow using Doppler ultrasound (DU) and strain gauge plethysmography (SGP) were compared in 10 normal volunteers at rest and during exercise. To evaluate the effect of strain gauge positioning, results of measurements made under four different combinations of cuff/strain gauge placement were compared in 15 other volunteers. The correlation of the limb blood flow obtained using the two methods at rest and exercise was 0.57 and 0.62 and the limits of agreement (d +/- 2SD) were 0.40 +/- 2.49 and -0.86 +/- 5.22 ml 100 ml-1 tissue min-1 at rest and on exercise, respectively. Results obtained using SGP were more reproducible (Coef. repeat. 0.45 vs. 0.94 ml 100 ml-1 tissue min-1, for SGP and DU, respectively). The various combinations of cuff/strain gauge positioning showed a tendency to over-read when the latter was placed on the thigh, but were not significantly different (P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)