The influence of COVID-19 on routine hematological parameters of hospitalized patients.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) was first discovered in Wuhan, China, in December 2019, and soon spread around the entire world. As no effective treatment is known, prediction of disease severity is very important in order to estimate a patients outcome. Aim of this study was to evaluate routine hematology parameters in time after admission. METHODS: Data from routine blood analyses from confirmed COVID-19 cases admitted to the University Hospital of Leuven in Belgium were collected. COVID-19 patients (n = 197) were assigned to three groups: a 'non-ICU' group, a 'ICU' group and a 'deceased' group. A control group of 60 Influenza A (non-COVID-19) patients was also included. The parameters evaluated were platelet count (PLT, 109/L), hemoglobin concentration (Hb, g/dL), leukocyte count (LEU, 109/L), neutrophil count (NEU, %), eosinophil count (EO, %), lymphocyte count (LYM, %) and monocyte count (MONO, %). RESULTS: Deceased COVID-19 patients had significant lower platelet count, higher leukocyte/neutrophil count, and lower eosinophil/lymphocyte/monocyte count compared to recovered patients. Especially lymphocyte count showed important differences; they were significantly lower between day 9 and 12 after admission making this time window important in predicting clinical worsening of a patient. CONCLUSION: Patients with COVID-19 with poor outcome showed significant differences in results of routine hematological parameters compared with patients that recovered. Especially lymphocyte count can be helpful in the prediction of a patients outcome.

A perinatal stress calculator for the neonatal intensive care unit: an unobtrusive approach.

OBJECTIVE: Early experience of pain and stress in the neonatal intensive care unit is known to have an effect on the neurodevelopment of the infant. However, an automated method to quantify the procedural pain or perinatal stress in premature patients does not exist. APPROACH: In the current study, EEG and ECG data were collected for more than 3 hours from 136 patients in order to quantify stress exposure. Specifically, features extracted from the EEG and heart-rate variability in both quiet and non-quiet sleep segments were used to develop a subspace linear-discriminant analysis stress classifier. MAIN RESULTS: The main novelty of the study lies in the absence of intrusive methods or pain elicitation protocols to develop the stress classifier. Three main findings can be reported. First, we developed different stress classifiers for the different age groups and stress intensities, obtaining an area under the curve in the range [0.78-0.93] for non-quiet sleep and [0.77-0.96] for quiet sleep. Second, a dysmature EEG was found in patients under stress. Third, an enhanced cortical connectivity and increased brain-heart communication was correlated with a higher stress load, while the autonomic activity did not seem to be associated to stress exposure. SIGNIFICANCE: The results shed a light on the pain and stress processing in preterm neonates, suggesting that software tools to investigate dysmature EEG might be helpful to assess stress load in premature patients. These results could be the foundation to assess the impact of stress on infants' development and to tune preventive care.

Integrating risk profiles for disease progression in the treatment choice for patients with lower urinary tract symptoms/benign prostatic hyperplasia: a combined analysis of external evidence and clinical expertise.

The RAND appropriateness method was used to explore the relevance of risk factors for disease progression in the treatment choice for patients with lower urinary tract symptoms/benign prostatic hyperplasia (LUTS/BPH). A total of, 12 international experts assessed the appropriateness of various treatments for 243 risk profiles. Highest appropriateness rates were found for alpha1-adrenoceptor antagonists (68% of profiles) and combination therapy (46%). A large prostate volume was the dominant argument in favour of 5alpha-reductase inhibitors and combination therapy, but was irrelevant for the choice of surgery. Considerable postvoid residual, severe symptoms and poor maximum flow rate were the most important factors in favour of surgery.

Risk factors for disease progression in patients with lower urinary tract symptoms/benign prostatic hyperplasia (LUTS/BPH): a systematic analysis of expert opinion.

Disease progression has become an important issue for the management of lower urinary tract symptoms/benign prostatic hyperplasia (LUTS/BPH). Although several risk factors have been identified, no specific patient risk profiles have been established that can be useful in the day-to-day management of LUTS/BPH. In this study, an international panel of urologists developed a risk classification based on the attribution of a risk score to 243 unique patient profiles. From the perspective of clinical decision making, it was concluded that postvoid residual, symptom severity and maximum flow rate are the most relevant determinants of the risk of disease progression.

Gastro-protective treatment in patients using NSAIDs. Development of appropriateness criteria by a multidisciplinary expert panel.

OBJECTIVE: To refine the appropriate indications for gastro-protective treatment in patients using non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: Using the RAND Appropriateness Method, a multidisciplinary expert panel was asked to rate the appropriateness of gastro-protection for 4608 different cases. Logistic regression was used to construct a decision framework to identify patients for whom gastro-protective medication should be considered. RESULTS: Complete consensus existed on the appropriate use of gastro-protection in patients with a history of (un)complicated peptic ulcer. For other patients. agreement was found for 39% over 1536 cases. Logistic regression demonstrated strong consistency of the panel ratings (Hosmer Lemeshow coefficient 0.92), allowing the development of a comprehensive decision support model. CONCLUSIONS: Using the RAND Appropriateness Method, we were able to develop a clear and internally consistent decision framework for the appropriate use of gastro-protection in patients taking NSAIDs. The validity of this model should be tested in further studies and practice.

The appropriate management of symptomatic gastro-oesophageal reflux disease (GORD) in primary care: a systematic analysis of expert opinion.

OBJECTIVE: To explore the appropriate indications for endoscopy and short-term anti-secretory treatment in patients with symptoms of gastro-oesophageal reflux disease (GORD). METHODS: The RAND Appropriateness Method (RAM) was used to systematically investigate the opinions of an expert panel (6 gastroenterologists and 6 general practitioners) on the appropriateness of either endoscopy or short-term medication for 768 different patient scenarios (cases). Each case was defined by the unique combination of diagnostic characteristics considered to be relevant in treatment choice. Panel members firstly individually rated the appropriateness of all indications using a 9-point scale (9 = extremely appropriate, 1 = extremely inappropriate). Subsequently, the panel discussed the results and re-rated some of the indications. Based on the median score and agreement figures, the individual ratings were converted to panel statements (appropriate, inappropriate, and uncertain) for each of the indications. Logistic regression was used to study the relationship between diagnostic characteristics and panel outcomes. RESULTS: Disagreement was seen in only 18% of cases. Statistical analysis revealed consistent patterns that determined the panel judgements. The most pronounced patterns and regression results were used to indicate situations in which either medication or referral was considered appropriate by the panel. CONCLUSION: The RAND panel method proved to be useful in the systematic analysis of expert opinion on the appropriate management of symptomatic GORD. Nevertheless, as the recommendations still reflect the subjective opinion of the panel members, their validity and usefulness for daily practice should be the subject of further investigations.

A comparison of targeting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients.

Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targeting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of 131I-MIBG (110 MBq) and with 131I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with 131I-chCE7, the subcutaneous tumours nearly disappeared; treatment with 131I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with 131I-mAb chCE7 and of 24 days with 131I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by 131I-chCE7 compared with 131I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with 131I-MIBG and 131I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. 131I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not accumulate 131I-MIBG, and the antibody may hold potential for radioimmunotherapy, either by itself or in combination with 131I-MIBG.

A comparison of targetting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients.

Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent (131)I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targetting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of (131)I-MIBG (110 MBq) and with (131)I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with (131)I-chCE7, the subcutaneous tumours nearly disappeared; treatment with (131)I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with (131)I-mAb chCE7 and of 24 days with (131)I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by (131)I-chCE7 compared with (131)I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with (131)I-MIBG and (131)I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. (131)I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not accumulate (131)I-MIBG, and the antibody may hold potential for radioimmunotherapy, either by itself or in combination with (131)I-MIBG.

Targeting of meta-iodobenzylguanidine to SK-N-SH human neuroblastoma xenografts: tissue distribution, metabolism and therapeutic efficacy.

The clinical results of [(131)I]meta-iodobenzylguanidine (MIBG)-targeted radiotherapy in neuroblastoma patients is highly variable. To assess the therapeutic potential of [(131)I]MIBG, we used the SK-N-SH human neuroblastoma, xenografted in nude mice. The model was first characterized for basic parameters of MIBG handling in the host species. This demonstrated the presence of both strain- and nu/nu mutation-related differences in [(131)I]MIBG biodistribution. Fecal and urinary clearance rates of [(131)I]MIBG in mice roughly resemble those in humans, but mice metabolize MIBG more extensively. In both species, enzymatic deiodination in vivo was not an important metabolic route. Therapy with increasing [(131)I]MIBG doses (25-92 MBq) given as single i.v. injections resulted in proportionally increasing specific growth delay values (tumor regrowth delay/doubling time) of 1 to 5. Using gamma-camera scintigraphy for non-invasive dosimetry, the corresponding calculated absorbed tumor radiation doses ranged from 2 to 11 Gy. We also compared the therapeutic effects of a single [(131)I]MIBG administration with those resulting from a more protracted exposure by fractionating the dose in 2 to 6 injections or with high dose rate external-beam irradiation. No therapeutic advantage of a fractionated schedule was observed, and 5.5 Gy delivered by low dose-rate [(131)I]MIBG endo-irradiation was equi-effective with 5.0 Gy X-rays. The SK-N-SH neuroblastoma xenograft model thus appears suitable to evaluate possible treatment improvements to reach full potential of MIBG radiotherapy.

[(131)I] and [(125)I] metaiodobenzylguanidine therapy in macroscopic and microscopic tumors: a comparative study in SK-N-SH human neuroblastoma and PC12 rat pheochromocytoma xenografts.

[(131)I]Metaiodobenzylguanidine ([(131)I]MIBG) targeted radiotherapy is effective in debulking childhood neuroblastoma. The high-energy beta-emitter [(131)I]MIBG is, however, not very well suited to treat submillimeter tumors. The [(125)I]MIBG emission is more fully absorbed in small target volumes and therefore advocated for treatment of microscopic neuroblastoma. We investigated whether i.v. [(125)I]MIBG can have a therapeutic advantage over i.v. [(131)I]MIBG in realistic animal models. We used BALB/c nu/nu mice, bearing neuroadrenergic xenografts which differ in MIBG handling, i.e., extragranular vs. granular MIBG storage in the SK-N-SH human neuroblastoma and PC12 rat pheochromocytoma, respectively. Groups of 4-9 animals were treated with 10-100 MBq radioiodinated MIBG. Responses were calibrated against the effect of 4-5 Gy of external beam X-rays. SUBCUTANEOUS XENOGRAFTS: Due to the more extensive MIBG accumulation, the estimated MIBG exposure of the PC12 tumor was nearly 20-fold higher compared with the SK-N-SH xenograft which corresponded with a marked, i.e., nine-fold increased tumor growth delay after radioiodinated MIBG therapy. Both xenografts were equally sensitive to high-dose rate local irradiation. In neuroblastoma as well as pheochromocytoma, the therapeutic efficacy of [(131)I]MIBG was 6 times higher compared to the [(125)I]MIBG which is in reasonable agreement with the reported "131-I over 125-I" ratio of approximately 9 for the calculated absorbed radiation doses per unit of radioactivity. Apparently, the neuroblastoma was not relatively more sensitive to the (ultra)short range emitter [(125)I]MIBG than the pheochromocytoma, indicating that its therapeutic efficacy is independent of the intracellular MIBG storage mode. MICROSCOPIC TUMORS: The pheochromocytoma model consisted of widespread disease after i.v. cell injection with survival as endpoint. For the neuroblastoma, we induced focal intrahepatic microscopic tumors by intrasplenic injection and evaluated total liver weights 26 days after therapy. Theoretically, the therapeutic potential of [(125)I]MIBG at the cellular level should be at least as high as [(131)I]MIBG, but we failed to show any effect of [(125)I]MIBG therapy in both models. In contrast, measurable responses were obtained with [(131)I]MIBG, but these were lower than in the s.c. tumors when related to the responses induced by external X-rays. In conclusion, [(131)I]MIBG is decreasingly effective in microscopic disease and can therefore not be curative as a single agent. Our results strongly argue against the clinical use of [(125)I]MIBG and indicate that conventional total body irradiation was superior to [(131)I]MIBG for microscopic neuroblastoma. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 312-325 (2000).

Bcl-2 family members in childhood acute lymphoblastic leukemia: relationships with features at presentation, in vitro and in vivo drug response and long-term clinical outcome.

We have found that, in addition to Bcl-2 and Bax, the expression levels of apoptosis inducers (Bad, Bak) and inhibitors (Bcl-xL, Mcl-1) were highly variable in blasts from 78 children with newly diagnosed acute lymphoblastic leukemia (ALL). The patients were enrolled in the national study ALL-7 of the Dutch Childhood Leukemia Study Group. In contrast to Bcl-2 that inversely correlated with %S-phase cells and WBC, and was lower in T than in B-lineage ALL, the Bcl-2 family members were not found to be associated with features at presentation. These expression levels were also compared with drug resistance in in vitro MTT (methyl-thiazol-tetrazolium) assays for prednisolone, vincristine and asparaginase in 46 children. Protein expression levels of the Bcl-2 family were not found to correlate with in vitroresistance to the individual drugs or the combined drug resistance profile. In addition, neither peripheral blast reduction after 1 week of prednisone monotherapy nor long-term disease-free interval or survival showed a correlation with protein expression. Our results indicate that the anti-proliferative function of Bcl-2 dominates its anti-apoptotic function in ALL, but neither Bcl-2 nor the Bcl-2 family members gained prognostic information in the risk-adapted protocol ALL-7.

Glucocorticoid induced apoptosis in leukemia.

Lymphoid and leukemic cells are uniquely sensitive to the lytic actions of glucocorticoid hormones which activate a programmed cell death in these cells. The response to glucocorticoids is sensitive to modulations at each step of hormone action: cellular uptake, binding and activation of cytosolic receptors, nuclear translocation and transcriptional activity of the activated receptor and the expression levels of pro- and anti-apoptotic genes. This review, based mainly on our studies with leukemic cells in tissue culture and on clinical observations in childhood acute lymphoblastic leukemia, summarizes the potential impact of these checkpoints in the treatment of this disease. In addition, we will discuss interventions that may reverse resistance or promote sensitivity to apoptosis of leukemic cells by glucocorticoid hormones.

Intensive treatment of children with acute lymphoblastic leukemia according to ALL-BFM-86 without cranial radiotherapy: results of Dutch Childhood Leukemia Study Group Protocol ALL-7 (1988-1991).

In The Netherlands from July 1988 to October 1991, children (0 to 16 years of age) with de novo acute lymphoblastic leukemia (ALL) were treated according to protocol ALL-7 of the Dutch Childhood Leukemia Study Group (DCLSG). In this protocol, chemotherapy and treatment stratification were identical to the ALL-BFM-86 protocol (Reiter et al, Blood 84:3122, 1994), but cranial irradiation was restricted to patients with initial central nervous system (CNS) involvement. Patients were stratified into 3 risk groups, based on leukemia cell mass and response to initial treatment: standard-risk group (SRG), risk group (RG), and experimental group (EG). As in ALL-BFM-86, a randomized study on late intensification (protocol S) was performed in RG patients, and during the study (since October 1990), early reinduction treatment (protocol II) was introduced for SRG patients. Treatment duration for all patients was 18 months. Two hundred eighteen children entered the study: 74 SRG, 127 RG, and 17 EG patients. The overall complete remission (CR) rate was 98%. The 5-year event-free survival (EFS) for all DCLSG ALL-7 patients was 65. 3% (standard error [SE] 3.2%), which was significantly different from the 73% (SE 1%) 5-year EFS achieved in the ALL-BFM-86 study (P =.02, Z-test). However, restricting the analysis to SRG patients receiving protocol II with a total duration of treatment of 18 months, the 5-year EFS rates were 64.6% (SE 4.0%) and 67% (SE 4%), respectively, and no significant difference could be established (P =.67, Z-test). The 5-year EFS rates for SRG, RG, and EG patients were 63.5% (SE 5.6%), 66.6% (SE 4.2%), and 63.3% (SE 12.0%), respectively. SRG patients receiving protocol II fared better than patients not receiving protocol II (5-year EFS 76.7% [SE 7.7] and 54. 5% [SE 7.5], respectively). No difference in 5-year EFS was observed in RG patients randomized to receive or not to receive late intensification with protocol S. The overall CNS relapse rate at 5 years was 5.5%. The incidence rate at 5 years was 11.4% in SRG patients not receiving protocol II, whereas no CNS relapses occurred in SRG patients receiving protocol II. Six children died in first complete remission and 2 children developed a second malignancy (thyroid carcinoma and acute nonlymphoblastic leukemia). Systemic high-dose methotrexate (MTX) and intrathecal chemotherapy is a safe and effective method of CNS prophylaxis in the context of BFM-oriented treatment for all children with ALL, regardless of the risk group (with the possible exception of T-ALL patients with high white blood cell counts). The results of the DCLSG ALL-7 study confirm those of the ALL-BFM-86 study showing that early reinduction with protocol II is essential in the treatment of SRG patients and that late intensification with protocol S does not improve the prognosis for RG patients.

Bioavailability and toxicity after oral administration of m-iodobenzylguanidine (MIBG).

meta-iodobenzylguanidine (MIBG) radiolabelled with iodine-131 is used for diagnosis and treatment of neuroadrenergic neoplasms such as phaeochromocytoma and neuroblastoma. In addition, non-radiolabelled MIBG, administered i.v., is used in several clinical studies. These include palliation of the carcinoid syndrome, in which MIBG proved to be effective in 60% of the patients. Oral MIBG administration might be convenient to maintain palliation and possibly improve the percentage of responders. We have, therefore, investigated the feasibility of oral administration of MIBG in an animal model. Orally administered MIBG demonstrated a bioavailability of 59%, with a maximal tolerated dose of 60 mg kg(-1). The first and only toxicity encountered was a decrease in renal function, measured by a reduced clearance of [51Cr]EDTA and accompanied by histological tubular damage. Repeated MIBG administration of 40 mg kg(-1) for 5 sequential days or of 20 mg kg(-1) for two courses of 5 sequential days with a 2-day interval did not affect renal clearance and was not accompanied by histological abnormalities in kidney, stomach, intestines, liver, heart, lungs, thymus, salivary glands and testes. Because of a sufficient bioavailability in absence of gastrointestinal toxicity, MIBG is considered suitable for further clinical investigation of repeated oral administration in patients.

Potentiation of anti-cancer drug activity at low intratumoral pH induced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) and its analogue benzylguanidine (BG).

Tumour-selective acidification is of potential interest for enhanced therapeutic gain of pH sensitive drugs. In this study, we investigated the feasibility of a tumour-selective reduction of the extracellular and intracellular pH and their effect on the tumour response of selected anti-cancer drugs. In an in vitro L1210 leukaemic cell model, we confirmed enhanced cytotoxicity of chlorambucil at low extracellular pH conditions. In contrast, the alkylating drugs melphalan and cisplatin, and bioreductive agents mitomycin C and its derivative EO9, required low intracellular pH conditions for enhanced activation. Furthermore, a strong and pH-independent synergism was observed between the pH-equilibrating drug nigericin and melphalan, of which the mechanism is unclear. In radiation-induced fibrosarcoma (RIF-1) tumour-bearing mice, the extracellular pH was reduced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) or its analogue benzylguanidine (BG) plus glucose. To simultaneously reduce the intracellular pH, MIBG plus glucose were combined with the ionophore nigericin or the Na+/H+ exchanger inhibitor amiloride and the Na+-dependent HCO3-/Cl- exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). Biochemical studies confirmed an effective reduction of the extracellular pH to approximately 6.2, and anti-tumour responses to the interventions indicated a simultaneous reduction of the intracellular pH below 6.6 for at least 3 h. Combined reduction of extra- and intracellular tumour pH with melphalan increased the tumour regrowth time to 200% of the pretreatment volume from 5.7 +/- 0.6 days for melphalan alone to 8.1 +/- 0.7 days with pH manipulation (P < 0.05). Mitomycin C related tumour growth delay was enhanced by the combined interventions from 3.8 +/- 0.5 to 5.2 +/- 0.5 days (P < 0.05), but only in tumours of relatively large sizes. The interventions were non-toxic alone or in combination with the anti-cancer drugs and did not affect melphalan biodistribution. In conclusion, we have developed non-toxic interventions for sustained and selective reduction of extra- and intracellular tumour pH which potentiated the tumour responses to selected anti-cancer drugs.

Signalling steps in apoptosis by ether lipids.

We have investigated the mechanisms of induction of apoptosis by the antineoplastic ether lipid ET-18-OCH3 (ALP) in sensitive S49wt mouse lymphoma cells and ALP-resistant S49ar variants, both with wild-type p53, and in related L1210 cells with mutated p53. Ether lipid-resistant S49ar cells were cross-resistant to extracellular stress factors (cold shock, heat shock, H2O2, dimethylsulfoxide) and to radiation-induced apoptosis but not to physiological apoptotic signals (dexamethasone, growth factor deprivation, thapsigargin, C2-ceramide) and expressed similar levels of the apoptosis-regulating proteins Bcl-2, Bcl-X, Bax, Bad and Bak as did the parent S49wt cells. The uptake of [3H]-ALP was strongly reduced in the stress-resistant cells but this was not associated with significant differences in membrane cholesterol:phospholipid content nor in membrane microviscosity. In S49ar cells the activity of the antioxidant enzyme glutathione peroxidase (GSH-Px) was increased 4-fold and depletion of glutathione with the drug L-buthionine-S-R-sulfoximine (L-BSO) lowered the resistance of S49ar cells to ALP, stress factors and ionising radiation. The results indicate that ether lipids induce apoptosis by imposing a special form of physico-chemical stress, mediated by reactive oxygen species but independent of p53 status. The capacity of glutathione-dependent anti-oxidant defence appeared an important and shared determinant of the sensitivity to ether lipids, several types of extracellular stress and ionising radiation.

Mutational analysis of Bax and Bcl-2 in childhood acute lymphoblastic leukaemia.

In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis-regulating proteins Bax and Bcl-2, but the molecular basis for this variation is unknown. Point-mutations in bax have been reported in cell lines derived from haematological malignancies. Frameshift mutations, which result in reduced Bax levels, have also been found in colon cancer of the microsatellite mutator phenotype. Bcl-2 overexpression, or gain of function mutations in the open reading frame (ORF) or in the translational repressor, the upstream ORF(uORF) of bcl-2, might also be important in deregulating its function or expression. We have therefore analyzed 21 bone marrow aspirates from untreated childhood acute lymphoblastic leukaemia and 2 from myeloid leukaemia for mutations in box and bcl-2. DNA sequence analysis of the ORFs of bax and bcl-2 and of the uORF of bcl-2 revealed no mutations, despite the large range in expression levels. Thus, mutations within the (u)ORFs of bax and bcl-2 that (in)activate or deregulate Bax and Bcl-2 are infrequent in primary childhood acute leukaemia and do not play a major role in regulation of the encoded proteins in this disease.

Renal toxicity of the neuron-blocking and mitochondriotropic agent m-iodobenzylguanidine.

meta-Iodobenzylguanidine (MIBG) is a multipotent drug used in its radiolabeled form as a tumor-seeking radiopharmaceutical in the diagnosis and treatment of pheochromocytoma and neuroblastoma. Nonradiolabeled MIBG has also proved to be effective in the palliation of carcinoid syndromes and, on a predosing schedule, in enhancing the relative tumor uptake of a subsequent [131I]-MIBG dose in tumors of neuroadrenergic origin. In addition, MIBG is under investigation as an inhibitor of mitochondrial respiration and, as such, for its use in tumor-specific acidification. In this report we describe the side effects of nonradiolabeled MIBG on kidney function in mice. High doses of MIBG (40 mg/kg) reduced renal blood perfusion as measured by 86Rb distribution by 50%, which could be antagonized by the bioamine receptor blockers prazosin and cyproheptadine. MIBG also induced reversible renal damage as evidenced from a decrease in [51Cr]-ethylenediaminetetraacetic acid (EDTA) clearance and from histological damage, which was most pronounced in the distal tubuli. These effects were unrelated to reduced perfusion, however, and could not be antagonized by bioamine receptor blockers, Ca2+-channel blockers, or diuretics. Clearance effects of MIBG were mimicked by N-nitro-L-arginine methyl ester (L-NAME), a known inhibitor of nitric oxide synthase (NOS), and MIBG itself (100 microM) also inhibited NOS in vitro, suggesting that NOS inhibition by MIBG may have contributed to the observed reduction in renal clearance. The MIBG analog benzylguanidine (BG), which is equipotent in terms of mitochondrial inhibition, did not affect renal clearance, thus excluding mitochondrial inhibition as the main mechanism of MIBG-induced damage. MIBG, however, was much more cytotoxic than BG to kidney tubular cells in primary cultures. Although the renal effects of high-dose MIBG were reversible, alterations in the pharmacokinetics of concomitant medications by a temporary reduction in renal function should be taken into account in its clinical application.