Induction of cell stress through gene transfer of an engineered heat shock transcription factor enhances tumor immunogenicity.

Heat shock protein expression and release is closely associated with immunogenic forms of cell death. We show that activation of the stress response within tumor cells during cell death, using an engineered form of the heat shock transcription factor, leads to an immunogenic death. Cells dying through 'stressful death' show decreased phagocytosis by macrophages in vitro. Moreover, cells expressing heat shock proteins during cell death are significantly more protective against subsequent tumor challenge. These data demonstrate the utility of activating cellular stress programs over the course of cytotoxic therapies to enhance immune responses to dying cells.

Macrophages orchestrate the immune response to tumor cell death.

The mechanisms by which the immune system distinguishes normal developmental cell death from pathological immunogenic cell killing are central to effective cancer immunotherapy. Using HSVtk suicide gene therapy, we showed that macrophages can distinguish between tumor cells dying through classical apoptosis and tumor cells engineered to die through nonapoptotic mechanisms, resulting in secretion of either immunosuppressive cytokines (interleukin 10 and transforming growth factor beta) or inflammatory cytokines (tumor necrosis factor alpha or interleukin 1beta), respectively. Additionally heat shock protein 70 acts as one component of a bimodal alarm signal that activates macrophages in the presence of stressful, immunogenic tumor cell killing. These differential responses of macrophages can also be used to vaccinate mice against tumor challenge, using adoptive transfer, as well as to cure mice of established tumors.

Long-term neurobehavioral outcomes in children with neuroblastoma and opsoclonus-myoclonus-ataxia syndrome: relationship to MRI findings and anti-neuronal antibodies.

OBJECTIVES: Opsoclonus-myoclonus-ataxia (OMA) syndrome affects 2% to 3% of patients with neuroblastoma. This study examined relationships between long-term neurobehavioral outcomes and potential biologic markers of OMA, including chronic changes on magnetic resonance imaging (MRI) brain scanning and prevalence of late antineuronal antibodies. STUDY DESIGN: Children with neuroblastoma and OMA were identified through medical record review of patients treated at the University of California at San Francisco Medical Center from 1979 to 1999. Eleven patients with a mean follow-up time of 7.6 years underwent standard neurologic, neurocognitive, developmental/behavioral, and academic assessments. Consenting patients underwent MRI brain scanning and a blood draw. Sera were analyzed for the presence of antineuronal immunoreactivity. RESULTS: Two (18%) patients had no observed neurologic abnormalities, 7 (64%) demonstrated mild deficits, and 2 (18%) had severe neurologic deficits. However, on neurocognitive, behavioral, and academic assessments, 6 (55%) children performed within the average range, 1 (9%) was moderately below average and 4 (36%) had severe cognitive and behavioral deficiencies. Brain MRI in 5 of 5 patients was notable for cerebellar atrophy without supratentorial involvement. Antineuronal activity was detected in sera of 0 of 10 children at follow-up. CONCLUSIONS: Certain patients with neuroblastoma associated OMA may achieve average-range neurobehavioral function in spite of residual neurologic abnormalities, with suggestion of continued improvement over time. Late cerebellar atrophy appears to be a common finding regardless of neurologic outcome, whereas antineuronal immune reactivity does not appear to be a long-term feature of OMA.

A multiple-hospital anaesthetic problem register: establishment of a regionally organized system for facilitated reporting of potentially recurring anaesthetic-related problems.

A regionally organized system aiming to facilitate reporting and retrieval of information about potentially recurring anaesthetic-related problems has been established, covering 20 separate hospitals. Components of the system include a reporting package to facilitate use by anaesthetists in busy clinical practice; centralized clerical support; supervision by anaesthetists; reports and laminated cards supplied to the patient; and a permanently accessible database. A new classification system for difficulties in airway management has been developed as part of the system. After initial establishment, the system has been utilized by a broad cross-section of anaesthetists in the region. The first 350 reports are described. The reporting rate is approximately 0.3% of all anaesthetics given in the region. We believe the success of this system has been primarily due to features aiming to facilitate reporting, "local" ownership and supervision by clinical anaesthetists.

Intraoperative metabolic monitoring of the heart: I. Clinical assessment of coronary sinus metabolites.

Numerous clinical studies have corroborated the ability of intraoperative sampling of coronary sinus blood to measure changes in myocardial metabolism induced by ischemia and reperfusion. Among other changes, cardiac arrest induces a period of obligate myocardial lactate production that persists for an indeterminate amount of time after reperfusion. Coronary sinus lactate assays have been established as a standard method to compare various myocardial protection strategies. Current methodology requires detailed sample processing, precluding real-time feedback in the operating room. Newer devices hold promise in allowing the online assessment of myocardial metabolism; however, these methods await precise validation.

Multi-photon microscopy in the evaluation of human saphenous vein.

BACKGROUND: The use of conventional fluorescence microscopy to image biological systems at the cellular level is limited by its inability to spatially resolve thick tissues. We have applied the technique of multi-photon fluorescence microscopy to study the structure and function of endothelial cells in living human saphenous vein taken from patients undergoing coronary artery bypass surgery. MATERIALS AND METHODS: Vein segments were preserved for 1-4 h to determine the temporal effects of storage. The effect of pH on endothelial and smooth muscle cell viability was examined by storing segments at pH 6.0, 7.4, and 8.0. Calcein-mediated green fluorescence and ethidium homodimer-mediated red fluorescence were used to differentiate cell viability. Increases in diaminofluorescein fluorescence were used to measure bradykinin activation of endothelial nitric oxide synthase (eNOS) with or without N-nitro-l-arginine (L-NNA). Multi-photon imaging was performed with the BioRad MRC1024ES system. RESULTS: Successful imaging of endothelial and smooth muscle cells of vein segments was achieved. Cell viability was well preserved up to 3 h of storage but dramatically decreased after 4 h. Cell viability was maintained at pH 7.4, diminished at pH 8.0, and was completely lost at pH 6.0. A two- to threefold increase in eNOS activity was observed upon activation by bradykinin which was completely inhibited in L-NNA-treated samples. CONCLUSIONS: We have demonstrated the successful application of multi-photon microscopy in imaging and quantifying nitric oxide production and cell viability under various storage conditions in human saphenous veins. This imaging technique allows for the functional imaging of cellular processes and may have diagnostic potential in cardiovascular surgery for patients undergoing bypass operations.

Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy.

BACKGROUND: The childhood-onset cerebral form of X-linked adrenoleukodystrophy, a demyelinating disorder of the central nervous system, leads to a vegetative state and death within 3-5 years once clinical symptoms are detectable. The hypothesis to be tested was whether bone-marrow transplantation can over an extended period of time halt the inexorable progressive demyelination and neurological deterioration. METHODS: 12 patients with childhood onset of cerebral X-linked adrenoleukodystrophy have been followed for 5-10 years after bone-marrow transplantation. Magnetic resonance imaging (MRI), neurological, neuropsychological, electrophysiological, and plasma very-long-chain fatty acid (VLCFA) measurements were used to evaluate the effect of this treatment. FINDINGS: MRI showed complete reversal of abnormalities in two patients and improvement in one. One patient showed no change from baseline to last follow-up. All eight patients who showed an initial period of continued demyelination stabilised and remained unchanged thereafter. Motor function remained normal or improved after bone-marrow transplantation in ten patients. Verbal intelligence remained within the normal range for 11 patients. Performance (non-verbal) abilities were improved or were stable in seven patients. Decline in performance abilities followed by stability occurred in five patients. Plasma VLCFA concentrations decreased by 55% and remained slightly above the upper limits of normal. INTERPRETATION: 5-10-year follow-up of 12 patients with childhood-onset cerebral X-linked adrenoleukodystrophy shows the long-term beneficial effect of bone marrow transplantation when the procedure is done at an early stage of the disease.

Pretreatment with intralesional hyaluronidase prior to excision of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSPs) are an uncommon malignancy that commonly recur, but rarely metastasize. The origin of DFSPs is controversial; however, they stain with the progenitor marker CD34. DFSPs usually show increased stromal mucin, mainly hyaluronic acid (HA). HA increases cellular proliferation, delays differentiation and increases cellular motion. We evaluated the pretreatment of DFSPs with intralesional injections of hyaluronidase (HD) prior to the surgical excision. Five of nine cases of DFSPs were pretreated with HD. In HD-pretreated cases the margins for excision of the residual tumor were reduced. HD pretreatment also decreased CD34 staining and increased polarizable collagen in the residual tumor.

Continuous perfusion of donor hearts in the beating state extends preservation time and improves recovery of function.

OBJECTIVES: Improving methods of donor heart preservation may permit prolonged storage and remote procurement of cardiac allografts. We hypothesized that continuous, sanguineous perfusion of the donor heart in the beating, working state may prolong myocardial preservation. METHODS: We developed a portable perfusion apparatus for use in donor heart preservation. Contractile, metabolic, and vasomotor functions were monitored simultaneously in an isolated swine heart. The metabolic state was monitored by myocardial tissue pH. Vasomotor function was assessed in isolated coronary ring chambers. Hearts were randomized into 3 groups: group I (n = 5), cardioplegic arrest, 12-hour storage at 4 degrees C with modified Belzer solution, and 2-hour sanguineous reperfusion in the working state; group II (n = 6), 12-hour continuous perfusion in the beating working state, 30 minutes of arrest (to simulate re-implantation time), and 2 hours of reperfusion, as above; group III (n = 7), coronary ring control hearts. RESULTS: At 2 hours of reperfusion, left ventricular developed pressure in group II was higher than in group I (mean +/- standard deviation: 90 +/- 6 mm Hg, 53 +/- 15 mm Hg, P = .005). Significantly less myocardial edema was observed in group II than in group I (73% +/- 4%, 80% +/- 1% water content, P = .01). Significantly less myocardial acidosis was noted in group II than in group I during preservation (pH 7.3 +/- 0.01, 6.1 +/- 0.03, P < .001) and reperfusion (pH 7.3 +/- 0.008, 6.8 +/- 0.05, P < .001). Coronary endothelial vasomotor function was better preserved in group II than in group I as evidenced by dose-response relaxation of coronary rings to 10(-8) mol/L bradykinin (37%, 55% delta baseline, P = .01). CONCLUSION: This new method extends the current preservation limit and avoids time-dependent ischemic injury, thereby allowing for distant procurement of donor organs.

Psychosocial consequences of bone marrow transplantation in donor and nondonor siblings.

We investigated the psychosocial effects of bone marrow transplantation (BMT) on siblings of transplant recipients. We asked how donor siblings compared with nondonor siblings on quantitative measures of behavior, psychological distress, and sense of self. Participants included 44 siblings (21 donors and 23 nondonors, ages 6-18 yr) of surviving pediatric BMT patients. On self-report measures, donors reported significantly more anxiety and lower self-esteem than nondonors. On teacher-rated scales, donors showed significantly more adaptive skills in school. On these same scales, nondonors showed significantly more school problems than donors. One-third of the siblings in each group reported a moderate level of post-traumatic stress reaction. Exploratory multiple regression analyses point to factors that might influence sibling adjustment and suggest counseling strategies and avenues for future research.

Cognitive and adaptive behavior 1 and 3 years following bone marrow transplantation.

Children receiving a bone marrow transplant (BMT) are at risk for neuropsychological late effects because of potentially neurotoxic chemotherapy and total body irradiation. The goal of this study was to prospectively and longitudinally assess the intellectual and adaptive functioning of children receiving a BMT. This study examined 67 children whose development was evaluated at baseline prior to BMT and at 1 year follow-up. Mean age at BMT was 45 months. Repeated-measures ANOVA indicated a significant decline in IQ between baseline and the 1 year follow-up evaluation. Multivariate and exploratory univariate analyses examined the potential influence of diagnosis, treatment regimen, cranial radiation dose, age at time of transplant, and sex of child but none of these independent variables predicted outcome. Twenty-six children (mean age at BMT of 28.4 months) were also given developmental evaluations 3 years post-BMT. Although IQ at the 1 year follow-up was significantly lower than baseline, no further changes were evident at the 3 year follow-up evaluation. Scores on the Vineland Adaptive Behavior Scales also dropped significantly between baseline and the 1 year follow-up, but did not change between the 1 year and 3 year evaluations.

Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome.

Long-term survival and improved neuropsychological function have occurred in selected children with Hurler syndrome (MPS I H) after successful engraftment with genotypically matched sibling bone marrow transplantation (BMT). However, because few children have HLA-identical siblings, the feasibility of unrelated donor (URD) BMT as a vehicle for adoptive enzyme therapy was evaluated in this retrospective study. Forty consecutive children (median, 1.7 years; range, 0.9 to 3.2 years) with MPS I H received high-dose chemotherapy with or without radiation followed by BMT between January 27, 1989 and May 13, 1994. Twenty-five of the 40 patients initially engrafted. An estimated 49% of patients are alive at 2 years, 63% alloengrafted and 37% autoengrafted. The probability of grade II to IV acute graft-versus-host disease (GVHD) was 30%, and the probability of extensive chronic GVHD was 18%. Eleven patients received a second URD BMT because of graft rejection or failure. Of the 20 survivors, 13 children have complete donor engraftment, two children have mixed chimeric grafts, and five children have autologous marrow recovery. The BM cell dose was correlated with both donor engraftment and survival. Thirteen of 27 evaluable patients were engrafted at 1 year following URD BMT. Neither T-lymphocyte depletion (TLD) of the bone marrow nor irradiation appeared to influence the likelihood of engraftment. Ten of 16 patients alive at 1 year who received a BM cell dose greater than or equal to 3.5 x 10(8) cells/kg engrafted, and 62% are estimated to be alive at 3 years. In contrast, only 3 of 11 patients receiving less than 3.5 x 10(8) cells/kg engrafted, and 24% are estimated to be alive at 3 years (P = .05). The mental developmental index (MDI) was assessed before BMT. Both baseline and post-BMT neuropsychological data were available for 11 engrafted survivors. Eight children with a baseline MDI greater than 70 have undergone URD BMT (median age, 1.5 years; range, 1.0 to 2.4 years). Of these, two children have had BMT too recently for developmental follow-up. Of the remaining six, none has shown any decline in age equivalent scores. Four children are acquiring skills at a pace equal to or slightly below their same age peers; two children have shown a plateau in learning or extreme slowing in their learning process. For children with a baseline MDI less than 70 (median age, 2.5 years; range, 0.9 to 2.9 years), post-BMT follow-up indicated that two children have shown deterioration in their developmental skills. The remaining three children are maintaining their skills and are adding to them at a highly variable rate. We conclude that MPS I H patients with a baseline MDI greater than 70 who are engrafted survivors following URD BMT can achieve a favorable long-term outcome and improved cognitive function. Future protocols must address the high risk of graft rejection or failure and the impact of GVHD in this patient population.

Toxicities of total-body irradiation for pediatric bone marrow transplantation.

PURPOSE: To determine the acute and late effects, including cognitive function, of total body irradiation (TBI) and chemotherapy for bone transplant (BMT) in children with immunodeficiency or hematologic disorders. METHODS AND MATERIALS: At UCSF, 15 children with immunodeficiency disorders and 58 children with leukemia received chemoradiotherapy between July 1982 and November 1993 and were evaluated for toxicity. Patients with severe combined immunodeficiency disorder (SCID) received 7 Gy TBI while leukemia patients received 12 Gy TBI. RESULTS: Eight immunodeficient patients (53%) are alive at 4 months to 11 years posttransplant. Acute toxicity was limited and treatment well tolerated. Most patients developed mild nausea and vomiting, skin rash, or erythema. Transient fever/chills, oral mucositis, and alopecia were noted in approximately 50% of patients. Seventy-three percent of all patients demonstrated acute liver dysfunction, but only four (27%) developed veno-occlusive disease. All children had decreased growth velocity but normal growth hormone levels. Other endocrinologic evaluations including adrenocorticotropic hormone (ACTH), cortisol, and thyroid hormones were normal. Only one evaluable girl had delayed puberty with late onset of secondary sexual characteristics. Neuropsychological testing demonstrated an intelligence quotient (IQ) reduction between the baseline and 1 year post-BMT, with some recovery at 3 years. Only one patient developed a clinically significant cataract. Thirteen percent of patients had chronic interstitial lung disease. Four children developed exostosis. Only 1 of the 15 children developed a second malignancy (acute myelogenous leukemia) at age 5, 51 months posttransplant for SCID. For patients with leukemia, similar toxicities were observed. Twenty-nine percent disease-free survival was noted with a mean follow-up of 4.7 years. Twenty-two percent had chronic interstitial lung disease and two patients were diagnosed with cataracts. Graft-vs.-host-disease (GVHD), pubertal development arrest, and delayed puberty were seen. One child developed papillary thyroid carcinoma, 49 months post-BMT. Similar neuropsychological testing decrements were also observed. CONCLUSION: Our experience suggests that intensive chemoradiotherapy, even at a young age, does not cause severe, acute, or late toxicities but does result in a small IQ decrement and the risk of secondary malignancy in children with long-term follow-up.

The developmental implications of prenatal and/or postnatal crack cocaine exposure in preschool children: a preliminary report.

Previous studies examining the development of prenatally cocaine-exposed children through 3 years of age have found no significant differences between exposed and control groups. This study explored the developmental correlates of prenatal and/or postnatal crack cocaine exposure in children between 4 and 6 years of age. Three groups were studied: Group 1, 18 prenatally-exposed children whose mothers continue to use crack; Group II, 28 children without prenatal exposure whose mothers presently use crack; and Group III, 28 children whose mothers never used crack. Mothers were street-recruited and were comparable in race and socioeconomic status. The three groups of children did not differ on neurological gross motor and expressive language measures. However, prenatally exposed children performed significantly worse than others on receptive language and visual motor drawing tests. Prenatal crack exposure predicted poor visual motor performance even after control for intrauterine alcohol and marijuana exposure, age, birth weight, and duration of maternal crack use.

Humanized antibody directed to the IL-2 receptor beta-chain prolongs primate cardiac allograft survival.

IL-2Rs are expressed by T cells activated in response to foreign histocompatibility Ags but not by normal cells. This difference in IL-2R expression is exploited by blockade of IL-2Rs to achieve immunosuppression. High affinity IL-2Rs involve three subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma. Murine Mik beta 1, a mAb that blocks IL-2 binding to IL-2R beta, was developed as an immunosuppressive agent. There was modest prolongation of cynomolgus cardiac allograft survival in animals treated with murine Mik beta 1 (mean survival 11.8 +/- 1.6 days compared with 8.2 +/- 0.4 days in untreated animals; p = 0.06). However, murine Mik beta 1 is ineffective in recruiting primate effector cells and is neutralized by monkey Abs directed toward the infused Ab. To circumvent these limitations, a humanized form of Mik beta 1, which is a largely human IgG1k Ab, except that murine hypervariable regions are retained, was developed. In vivo plasma survival of humanized Mik beta 1 was threefold longer than simultaneously administered murine Mik beta 1 (terminal t1/2, 104 +/- 10 h vs 37 +/- 2 h). Furthermore, humanized Mik beta 1 manifests Ab-dependent cellular cytotoxicity, an activity that is absent with the parental murine Mik beta 1. Graft survival was significantly prolonged by humanized Mik beta 1 treatment with survivals of 22, 22, 24, 27, 44, and > 300 days (p vs control < 0.01; p vs murine Mik beta 1 < 0.01). Survival was not prolonged further (p > 0.3) by the addition of humanized anti-Tac, which blocks interaction of IL-2 with IL-2R alpha subunits. There was no toxicity attributable to the use of Mik beta 1 Abs. Thus, humanized Mik beta 1 prolonged cardiac allograft survival in primates without toxicity and may be effective as an adjunct to standard immunosuppressive therapy.

Coronary arteriographic findings in black patients and risk markers for coronary artery disease.

Coronary arteriographic results are reported in 1535 black patients: 751 men (mean age 57 +/- 11) and 784 women (mean age 59 +/- 11). Among the black men 19%, 15%, 21%, and 4% had single-, double-, and triple-vessel and left main disease, respectively. Among the black women there were 12%, 10%, 15%, and 3% with similar involvement. Logistic regression models showed that most of the recognized risk factors were positively correlated with significant (at least one artery with > or = 50% stenosis) coronary disease, but a history of hypertension was not a significant independent predictor in either sex. ECG evidence of previous infarction increased the odds of detecting significant coronary disease by the greatest amount when controlling for other significant risk markers in women. In men both previous infarction and atypical pain (negative) were equally important. This study confirms but does not explain previous reports that have revealed less than expected angiographic evidence of significant coronary artery disease in black compared with white persons.

Nicardipine: myocardial protection in isolated working hearts.

The effectiveness of the calcium antagonist nicardipine in protecting the ischemic myocardium was evaluated using the hemodynamic recovery of isolated working rat hearts subjected to hyperkalemic cardiac arrest followed by ischemia at 37.5 degrees C and 10 degrees C. Rat hearts (n = 51) received 20 mL of cardioplegia and were subjected to 27 minutes of ischemia at 37.5 degrees C. Group A (control) did not receive nicardipine. Groups B through F received nicardipine in the cardioplegia with total doses ranging from 2 micrograms to 6 micrograms. Group A had 46% survival of ischemia, whereas groups C (3 micrograms) and D (4 micrograms) had survival rates of 88% and 100%, respectively (p less than 0.05). The recovery of aortic flow after ischemia was 35% in group A, compared with 76% in group B (2 micrograms) and 81% in group D (p less than 0.05). Group A had 49% postischemic recovery of cardiac output, whereas groups B and D had 82% and 85% recovery (p less than 0.05). The postischemic recovery of stroke volume was 48% in group A compared with 84% in group B, 87% in group D, and 73% in group E (5 micrograms) (p less than 0.05). Additional rats were exposed to 210 minutes of ischemia (n = 41) or 240 minutes of ischemia (n = 56) at 10 degrees C. Control groups did not receive nicardipine, whereas treatment groups received nicardipine in the cardioplegia with total doses ranging from 1.4 micrograms to 6.4 micrograms. There were no significant differences in the survival of ischemia or the recovery of function after ischemia at 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

A prospective study of cognitive functioning following low-dose cranial radiation for bone marrow transplantation.

This study prospectively examined the effects of low doses of cranial irradiation on psychological development in children. The subjects were 22 children receiving bone marrow transplantation. Fifteen children receiving cranial doses ranging from 350 to 1200 cGy as part of their conditioning regimen were contrasted to seven children who were not irradiated. Measures of intellectual and psychosocial development were administered to all subjects prior to transplant and again at one-year posttransplant. There were no decrements in psychological functioning at the one-year follow-up, regardless of the dose of cranial radiation received or the age at which radiation was administered. In addition, there were no significant differences in test scores relative to baseline in a smaller cohort of children followed up for 3 years. Although no significant between-group findings were found, examination of individual cases revealed a high degree of variability, with decrements in IQ of 10 points or more found in 7 children at the one-year follow-up. Findings suggest that doses less than 1500 cGy may prove to be relatively well tolerated with respect to long-term cognitive and psychosocial development, even in young children, although caution is urged in light of the limited length of follow-up.

Methylene blue guidance for simplified resection of a lung lesion.

A 60-year-old patient returned 1 year after right pneumonectomy with a new primary squamous cell carcinoma of the left lower lobe. Using fluoroscopic guidance, the lesion and the shortest track to the surface were marked by methylene blue preoperatively. The lesion was easily excised by wedge resection without the need for manipulation or deflation of the lung.