The use of biomarkers to stratify surgical care in women with ovarian cancer: Scientific Impact Paper No. 69 May 2022.

Biomarkers may offer unforeseen insights into clinical diagnosis, as well as the likely course and outcome of a condition. In this paper, the focus is on the use of biological molecules found in body fluids or tissues for diagnosis and prediction of outcome in ovarian cancer patients. In cancer care, biomarkers are being used to develop personalised treatment plans for patients based on the unique characteristics of their tumour. This tailoring of care can be used to pursue specific targets identified by biomarkers, or treat the patient according to specific tumour characteristics. Surgery is one of the core treatments for ovarian cancer, whether it is offered in primary surgery or following chemotherapy in delayed surgery. Biomarkers already exist to guide the treatment of tumours with chemotherapy, but very little research has determined the value of biomarkers in tailoring surgical care for ovarian cancer. Such research is required to identify new biomarkers and assess their effectiveness in a clinical setting as well as to help identify specific tumour types to guide surgery. Biomarkers could help to determine the success of removing the disease surgically, or help to identify tumour deposits that persist after chemotherapy. All of these aspects would improve current practice. This Scientific Impact Paper highlights research that may pave the way towards bespoke surgery according to the biological characteristics of a tumour and aid gynaecological oncologists to provide surgical treatment according to individual need, rather than a blanket approach for all.

Evaluating retinopathy of prematurity screening guidelines for 24- to 27-week gestational age infants.

OBJECTIVE: To determine whether current retinopathy of prematurity (ROP) screening guidelines adequately identify treatable ROP in a contemporary cohort of extremely low gestation infants. STUDY DESIGN: Data from the Surfactant, Positive Pressure, and Pulse Oximetry Randomized Trial were used. Inborn infants of 24 (0)/7 to 27 (6)/7 weeks gestational age (GA) with consent before delivery were enrolled in 2005 to 2009. Severe ROP (type 1 ROP or treatment with laser, cryotherapy or bevacizumab) or death was the primary outcome for the randomized trial. Examinations followed the then current AAP (American Academy of Pediatrics) screening recommendations, beginning by 31 to 33 weeks postmenstrual age (PMA). RESULT: One thousand three hundred and sixteen infants were enrolled in the trial. Nine hundred and ninety-seven of the 1121 who survived to first eye exam had final ROP outcome determined. One hundred and thirty-seven (14% of 997) met criteria for severe ROP and 128 (93%) of those had sufficient data (without missing or delayed exams) to determine age of onset of severe ROP. PMA at onset was 32.1 to 53.1 weeks. In this referral center cohort, 1.4% (14/997) developed severe ROP after discharge. CONCLUSION: Our contemporary data support the 2013 AAP screening guidelines for ROP for infants of 24 (0)/7 to 27 (6)/7 weeks GA. Some infants do not meet treatment criteria until after discharge home. Post-discharge follow-up of infants who are still at risk for severe ROP is crucial for timely detection and treatment.

Blood stream infection is associated with altered heptavalent pneumococcal conjugate vaccine immune responses in very low birth weight infants.

OBJECTIVE: Sepsis in older children and adults modifies immune system function. We compared serotype-specific antibody responses to heptavalent pneumococcal conjugate vaccine (PCV7) in very low birth weight infants (<1500 g,VLBWs) with and without blood stream infection (BSI) during their birth hospitalization. STUDY DESIGN: Retrospective analysis of prospectively collected data for the Neonatal Research Network study of PCV7 responses among VLBWs. Infants received PCV7 at 2, 4 and 6 months after birth with blood drawn 4 to 6 weeks after third dose. Serotype antibodies were compared between infants with or without a history of BSI. Regression models were constructed with BW groups and other confounding factors identified in the primary study. RESULT: In all, 244 infants completed the vaccine series and had serum antibody available; 82 had BSI. After adjustment, BSI was not associated with reduced odds of serum antibody 0.35 µg ml(-1). CONCLUSION: BSI was not associated with reduced odds of World Health Organization-defined protective PCV7 responses in VLBWs.

Efficacy of phototherapy devices and outcomes among extremely low birth weight infants: multi-center observational study.

OBJECTIVE: Evaluate the efficacy of phototherapy (PT) devices and the outcomes of extremely premature infants treated with those devices. STUDY DESIGN: This substudy of the National Institute of Child Health and Human Development Neonatal Research Network PT trial included 1404 infants treated with a single type of PT device during the first 24±12 h of treatment. The absolute (primary outcome) and relative decrease in total serum bilirubin (TSB) and other measures were evaluated. For infants treated with one PT type during the 2-week intervention period (n=1223), adjusted outcomes at discharge and 18 to 22 months corrected age were determined. RESULT: In the first 24 h, the adjusted absolute (mean (±s.d.)) and relative (%) decrease in TSB (mg dl(-1)) were: light-emitting diodes (LEDs) -2.2 (±3), -22%; Spotlights -1.7 (±2), -19%; Banks -1.3 (±3), -8%; Blankets -0.8 (±3), -1%; (P<0.0002). Some findings at 18 to 22 months differed between groups. CONCLUSION: LEDs achieved the greatest initial absolute reduction in TSB but were similar to Spots in the other performance measures. Long-term effects of PT devices in extremely premature infants deserve rigorous evaluation.

Does aggressive phototherapy increase mortality while decreasing profound impairment among the smallest and sickest newborns?

OBJECTIVE: Aggressive phototherapy (AgPT) is widely used and assumed to be safe and effective for even the most immature infants. We assessed whether the benefits and hazards for the smallest and sickest infants differed from those for other extremely low-birth-weight (ELBW; ≤ 1000 g) infants in our Neonatal Research Network trial, the only large trial of AgPT. STUDY DESIGN: ELBW infants (n=1974) were randomized to AgPT or conservative phototherapy at age 12 to 36 h. The effect of AgPT on outcomes (death, impairment, profound impairment, death or impairment (primary outcome), and death or profound impairment) at 18 to 22 months of corrected age was related to BW stratum (501 to 750 g; 751 to 1000 g) and baseline severity of illness using multilevel regression equations. The probability of benefit and of harm was directly assessed with Bayesian analyses. RESULT: Baseline illness severity was well characterized using mechanical ventilation and FiO(2) at 24 h age. Among mechanically ventilated infants ≤ 750 g BW (n=684), a reduction in impairment and in profound impairment was offset by higher mortality (P for interaction <0.05) with no significant effect on composite outcomes. Conservative Bayesian analyses of this subgroup identified a 99% (posterior) probability that AgPT increased mortality, a 97% probability that AgPT reduced impairment, and a 99% probability that AgPT reduced profound impairment. CONCLUSION: Findings from the only large trial of AgPT suggest that AgPT may increase mortality while reducing impairment and profound impairment among the smallest and sickest infants. New approaches to reduce their serum bilirubin need development and rigorous testing.

Influence of clinical status on the association between plasma total and unbound bilirubin and death or adverse neurodevelopmental outcomes in extremely low birth weight infants.

OBJECTIVES: To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18-22 months corrected age in extremely low birth weight infants. METHOD: Total plasma bilirubin and unbound bilirubin were measured in 1101 extremely low birth weight infants at 5 +/- 1 days of age. Clinical criteria were used to classify infants as clinically stable or unstable. Survivors were examined at 18-22 months corrected age by certified examiners. Outcome variables were death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death prior to follow-up. For all outcomes, the interaction between bilirubin variables and clinical status was assessed in logistic regression analyses adjusted for multiple risk factors. RESULTS: Regardless of clinical status, an increasing level of unbound bilirubin was associated with higher rates of death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss and death before follow-up. Total plasma bilirubin values were directly associated with death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death before follow-up in unstable infants, but not in stable infants. An inverse association between total plasma bilirubin and death or cerebral palsy was found in stable infants. CONCLUSIONS: In extremely low birth weight infants, clinical status at 5 days of age affects the association between total plasma bilirubin and death or adverse neurodevelopmental outcomes at 18-22 months of corrected age. An increasing level of UB is associated a higher risk of death or adverse neurodevelopmental outcomes regardless of clinical status. Increasing levels of total plasma bilirubin are directly associated with increasing risk of death or adverse neurodevelopmental outcomes in unstable, but not in stable infants.

Double wall versus single wall incubator for reducing heat loss in very low birth weight infants in incubators.

BACKGROUND: Studies have shown improved survival of newborn infants maintained in the thermoneutral range. The concept of an incubator with additional insulation, a double plexiglass wall, is appealing for very low birth weight infants as it may help to provide a thermoneutral environment. OBJECTIVES: To assess the effects of double walled incubator versus a single wall incubator on insensible water loss, rate of oxygen consumption, episodes of hypothermia, time to regain birth weight, duration of hospitalization and infant mortality in premature infants. SEARCH STRATEGY: The standard search strategy of the Cochrane Neonatal Review Group was used. This included searches of electronic databases: Oxford Database of Perinatal Trials, Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 1, 2006), MEDLINE (1966 - 2006), EMBASE, previous reviews including cross references, abstracts, conference and symposia proceedings, expert informants in all published languages, and CINAHL (1982 - 2006). SELECTION CRITERIA: Only studies using random or quasi-random methods of allocation were considered for this review. Eligible studies assessed at least one of the outcome variables identified as important to this topic. DATA COLLECTION AND ANALYSIS: Independent data extraction and quality assessment of included trials was conducted by the review authors. Data were analyzed using generic inverse variance methodology and weighted mean difference (WMD). Results are presented with 95% confidence intervals. Meta-analysis was undertaken using a fixed effect model. MAIN RESULTS: Three studies met the criteria. Four other studies were excluded, as they did not compare double versus single wall incubators (details of the studies are given in the included and excluded studies section). Double wall incubators have the advantage of decreasing heat loss, decreasing heat production and decreasing radiant heat loss when compared to single wall incubators. There is also the advantage of reduced oxygen consumption. A minimal increase in conductive heat loss was noted when compared to single wall incubators. All of these effects are small and do not support the proposition that double wall incubators have a beneficial effect on long term outcomes including mortality or the duration of hospitalization. AUTHORS' CONCLUSIONS: Although it appears that caring for extremely small infants in double wall incubators may theoretically result in shorter hospitalization and may have metabolic advantages, this review was unable to find any data in the literature to support or refute this hypothesis. The studies do not provide any evidence that the small decrease in heat loss improves clinical outcome. Therefore, the available data is insufficient to directly guide clinical practice.

Erythromycin for the prevention of chronic lung disease in intubated preterm infants at risk for, or colonized or infected with Ureaplasma urealyticum.

BACKGROUND: Controversy exists over whether or not Ureaplasma urealyticum colonization or infection of the respiratory tract contributes to the severity of chronic lung disease (CLD), a major cause of morbidity and mortality in preterm infants. OBJECTIVES: To evaluate the efficacy and safety of prophylactic or therapeutic erythromycin in preventing chronic lung disease in intubated preterm infants with unknown U. urealyticum status or proven positivity. SEARCH STRATEGY: Searches were done of MEDLINE (1966-June 9, 2003), EMBASE (1980-May 5, 2003), The Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 4, 2002), previous reviews including cross-references, and abstracts of conference proceedings (Pediatric Academic Societies 2000-2003, American Thoracic Society 2001-2003). There were no language restrictions. Expert informants were contacted. SELECTION CRITERIA: Randomized or quasi-randomized studies comparing either prophylactic or therapeutic administration of oral or intravenous erythromycin (regardless of dose and duration) versus no treatment or placebo among intubated preterm infants <37 weeks and <2500 grams with either unknown U. urealyticum status or proven positivity by culture or polymerase chain reaction. DATA COLLECTION AND ANALYSIS: Data were extracted by all of the authors independently and differences were resolved by consensus. Treatment effects for categorical outcomes were expressed as relative risk, with 95% confidence intervals. MAIN RESULTS: Two small controlled studies, both involving intubated babies <30 weeks gestation, were eligible for inclusion. Lyon 1998 tested prophylactic erythromycin in babies whose U. urealyticum status was unknown at the time of initiation of treatment. Jonsson 1998 tested erythromycin in babies known to be culture positive for U. urealyticum. Neither trial showed a statistically significant effect of erythromycin on CLD, death or the combined outcome CLD or death. Because the two studies differed importantly in their design, the results were not combined in meta-analyses. No adverse effects of a 7-10 day course of erythromycin were reported in either study. REVIEWER'S CONCLUSIONS: Current evidence does not demonstrate a reduction in CLD/death when intubated preterm infants are treated with erythromycin prophylactically before U. urealyticum culture/PCR results are known or when Ureaplasma colonized, intubated preterm infants are treated with erythromycin. However, a true benefit could easily have been missed with the small sample sizes in the two eligible studies. The studies were greatly underpowered to detect uncommon adverse effects such as pyloric stenosis. Additional controlled trials are required to determine whether antibiotic therapy of Ureaplasma reduces CLD and/or death in intubated preterm infants.

Both human SP-A1 and Sp-A2 genes are expressed in small and large intestine.

The human SP-A locus consists of two functional genes and one pseudogene, and SP-A is shown to play a role in local host defense and the regulation of inflammation in lung. Because the intestine, like the lung, is constantly exposed to foreign and potentially harmful substances, we investigated the hypothesis that both human SP-A genes are expressed in intestine. We demonstrate that both SP-A genes are expressed in human small and large intestine. The presence of SP-A mRNA in human intestine was detected by reverse transcription polymerase chain reaction (RT-PCR), Northern blot analysis, and immunohistochemistry. The size of intestinal SP-A mRNA is the same as that in human lung, but the level of expression, compared with that in the lung, is very low in both the small and large intestine. Immunohistochemical analysis revealed positive reactivity for SP-A in a subgroup of epithelial cells in the intestine. Expression of both SP-A1 and SP-A2 genes was established by gene-specific PCR amplification, PCR-based converted RFLP discrimination, and direct sequencing of RT-PCR products. We speculate that SP-A in the intestine plays a role in local host defense and inflammation.

Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors.

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the detection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a potent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed by flow cytometry, fluorescence microscopy, or a fluorescence plate reader. Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versatile and specific tool for detecting activated caspases in living cells.

Stathmo-apoptosis: arresting apoptosis by fluorochrome-labeled inhibitor of caspases.

Apoptosis, like mitosis, is a kinetic event. The entire duration of apoptosis, from its onset to total disintegration of the cell, is often short and may be of variable duration. The time-window through which individual apoptotic cells display their characteristic features that serve to identify them varies depending on: a) the assay that is used, b) the cell type, c) the nature of the inducer of apoptosis, and d) the environmental factors the cell is exposed to that may shorten or prolong apoptosis. Thus, because the apoptotic index (AI) does not accurately represent incidence of apoptosis it is desirable to estimate the rate of cell death in analogy to the cell birth rate which is assessed by the stathmo-kinetic approach by arresting cells in mitosis. In this study the fluorescent caspase inhibitor FAM-VAD-FMK was used for dual purposes: a) to arrest the process of apoptosis (stathmo-apoptosis), and b) to have the arrested cells labeled with fluorochrome. Apoptosis of HL-60 and MCF-7 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) and FAM-VAD-FMK was added at the same time as the inducer. While the cells become progressively labeled with FAM-VAD-FMK, their disintegration, loss of the phase-contrast and loss of the capability to bind the inhibitor, and in the case of MCF-7 cells, detachment from the slides, all were prevented for up to 48 h. The percentage of FAM-VAD-FMK labeled HL-60 cells was plotted as a function of time after addition of CPT and the rate of cell entrance to apoptosis was estimated from the slopes of the stathmo-apoptotic plot at different time after administration of CPT. The plot revealed the presence of two distinct subpopulations: during the initial 8 h of the treatment with CPT the cells of the first subpopulation, predominantly the S-phase cells, were entering apoptosis at a rate of about 7% of cells per hour. The remaining cells were stochastically entering apoptosis between 8 and 48 h at a rate 1% of cells per hour. The present approach offers a unique capability to accurately estimate the kinetics of cell transition to apoptosis, revealing the unbiased cumulative apoptotic index over a long time span after induction of apoptosis.

Surfactant regulation of host defense function in the lung: a question of balance.

Although the lung is protected by classic innate and adaptive immune mechanisms, another unique local immunoregulatory system involving pulmonary surfactant is described in this review. Normal surfactant inhibits many immune cell functions including proliferation resulting from various stimuli and production of reactive oxidants, inflammatory mediators, and some cell surface markers. The predominant surfactant lipids appear to be responsible for these suppressive effects. Conversely, surfactant proteins SP-A and SP-D stimulate many aspects of immune cell behavior. These proteins are collagenous lectins or collectins that bind to glycoconjugates on many pathogens, enhancing phagocytosis and killing in some cases. SP-A and SP-D stimulate chemotaxis and reactive oxidant generation, particularly in macrophages, although other cells are probably affected as well. In some cases, SP-A also stimulates the expression of cell surface markers and is involved in the stimulation of inflammatory mediators. Under normal conditions, the inhibitory effects of the lipid prevail, but the collectins may provide focal activation and stimulate immune cells at sites where they are needed. However, in some types of lung disease or after certain insults or exposures, the balance between these inhibitory and stimulatory influences may be disrupted and result in inflammatory injury.

Do urinary levels of vascular endothelial growth factor predict proliferative retinopathy?

PURPOSE: Vascular endothelial growth factor (VEGF) is elevated in the vitreous of patients with proliferative retinopathies (PR). Angiogenic factors like VEGF are elevated in the urine of subjects with cancers, including those distant from the genitourinary tract. We hypothesized that local increases in VEGF in the vitreous would be reflected in the urine of subjects with PR. METHODS: Urine samples were collected from adults with absent, mild, or severe (requiring laser photocoagulation) PR. VEGF was measured by enzyme linked immunosorbent assay. RESULTS: Of 42 subjects, 16 had no PR and 26 had PR (8 mild, 18 severe). Thirty subjects had diabetes mellitus; 24 of these had PR. Subjects with PR were older than controls. Subjects with PR tended to have higher urinary VEGF (median 123 pg/ml Cr, range 3--1738) than controls without PR (median 93 pg/ml Cr, range 2--200) (p = 0.08). None of 16 controls, but 11/15 subjects with PR had >200 mg VEGF/mg Cr (p = 0.003), yielding high specificity (100%), but poor sensitivity (42%) of elevated urinary VEGF for PR. Urinary VEGF was also modestly correlated with urinary protein excretion (r(2 ) = 0.23). Correction of VEGF values for urinary protein abrogated any correlation with PR. CONCLUSIONS: Urinary levels of VEGF are associated with PR, but this relationship may be caused by concurrent renal diseases that result in proteinuria and/or renal VEGF production. The insensitivity of the association may preclude its use in screening to avoid eye examinations.

Early light reduction for preventing retinopathy of prematurity in very low birth weight infants.

BACKGROUND: Retinopathy of prematurity (ROP) causes vision loss in many premature infants each year, despite the advances being made in treatment. In the search for ways to prevent the disease altogether, the exposure of the retina to bright ambient light following premature birth has been a natural hypothesis, since the premature infant normally would be in the dark in-utero environment. Several controlled studies have now addressed this theory. OBJECTIVES: To answer the question: "Among very low birth weight infants, what is the effect of reducing early environmental light exposure on the incidence of any "Acute ROP", or "Poor ROP Outcomes"? SEARCH STRATEGY: Searches were made of the Cochrane Neonatal Group Register of Controlled Trials, Medline, EMBASE, the Cochrane Library, previous reviews including cross references, abstracts, conference and symposia proceedings, and expert informants. The search terms used were [retrolental fibroplasia or retinopathy of prematurity] and [light or light/ae or lighting or lighting/ae or light/tu or lighting/st]. This search was updated as of November 2000. SELECTION CRITERIA: Randomized or quasi-randomized controlled trials that reduced light exposure to premature infants within the first 7 days following birth were considered for this review. DATA COLLECTION AND ANALYSIS: Data on clinical outcomes including any Acute ROP and Poor ROP Outcome were excerpted by both reviewers independently and consensus reached. Data analysis was conducted according to the standards of the Neonatal Cochrane Review Group. MAIN RESULTS: Data from four recent randomized trials, and one much older quasi-randomized trial failed to show any reduction in Acute ROP, or Poor ROP Outcome with the reduction of ambient light to premature infants' retinas. The number of infants studied to date allows 95% confidence that IF there were a true difference being missed, it would be smaller than 7 percentage points on a background of 54% of all infants under 2 kg developing ROP. REVIEWER'S CONCLUSIONS: Decreasing retinal ambient light exposure in premature infants is very unlikely to reduce the incidence of ROP.

D-Penicillamine for preventing retinopathy of prematurity in preterm infants.

BACKGROUND: Retinopathy of prematurity remains a common problem. A low rate of this disorder was unexpectedly observed among infants treated with intravenous d-penicillamine to prevent hyperbilirubinemia. This observation led to the investigation of its use to prevent retinopathy of prematurity. OBJECTIVES: To answer the question: Among very low birth weight infants, what is the effect of prophylactic administration of d-penicillamine on the incidence of acute ROP or severe ROP, and side effects including death? SEARCH STRATEGY: Searches were made of multiple electronic databases, previous reviews including cross references, abstracts, conference/symposia proceedings, and expert informants. The search was updated to November 2000. SELECTION CRITERIA: Randomized or quasi-randomized controlled trials that administered d-penicillamine to infants less than 2000g birth weight within the day following birth were considered relevant to this review. Additional case series were examined for potential side effects. DATA COLLECTION AND ANALYSIS: Data on clinical outcomes were excerpted by 3 reviewers independently, and consensus reached. Data analysis was conducted according to the standards of the Neonatal Cochrane Review Group. MAIN RESULTS: Two randomized trials on the effects on ROP were identified. When combined, they showed a significantly lower incidence of acute ROP in the treated infants, relative risk of 0.09, 95% CI [0.01,0.71]. Severe stages of ROP could not be analyzed. There was no effect on death rates, relative risk 0.99 95% CI [0.70,1.39]. No side effects were reported, and follow up at one year revealed no significant differences in spasticity or developmental delay, although there were more rehospitalizations among the controls. In other reports of using d-penicillamine in over 140 infants for hyperbilirubinemia, skin rashes were reported in 2 infants and one had vomiting that may have been related. REVIEWER'S CONCLUSIONS: D-penicillamine is unlikely to affect survival, and may reduce the incidence of acute ROP among survivors. Studies to date justify further investigation of this drug in a broader population; careful attention to possible side effects is needed.

Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry.

BACKGROUND: The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308-313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. METHODS: Apoptosis of human MCF-7, U-937, or HL-60 cells was induced by camptothecin (CPT) or tumor necrosis factor-alpha (TNF-alpha) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluorochromes was measured by LSC. RESULTS: Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was largely (> 90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. CONCLUSIONS: The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation.

Use of a modified ornithine decarboxylase promoter to achieve efficient c-MYC- or N-MYC-regulated protein expression.

One of the advantages of viral-directed enzyme prodrug therapy (VDEPT) is its potential for tumor-specific cytotoxicity. However, the viruses used to deliver cDNAs encoding prodrug-activating enzymes transduce normal cells as well as tumor cells, and several approaches to achieve tumor-specific expression of the delivered cDNAs are being investigated. One such approach is to regulate transcription of the prodrug-activating enzyme with a promoter that is preferentially activated by tumor cells. Published data suggest that the most promising transcription factor/promoter/enhancer combinations are those activated by a tumor-specific transcription factor to retain tumor cell specificity but that are equal in strength to nonspecific viral promoters in their ability to up-regulate target cDNAs. This report identifies MYC-responsive, modified ornithine decarboxylase (ODC) promoter/enhancer sequences that up-regulate target protein expression in tumor cells overexpressing either N-MYC or c-MYC protein. The most efficient of the four constructs assessed contained six additional CACGTG MYC binding sites 5' to the endogenous ODC promoter (R6ODC). Reporter assays with this chimeric promoter/enhancer regulating expression of chloramphenicol acetyltransferase demonstrated 50-250-fold more activity in MYC-expressing cells compared with similar assays with promoterless plasmids. The R6ODC regulatory sequence was approximately equivalent to the CMV promoter in inducing expression of the neomycin resistance gene in c-MYC-expressing SW480 and HT-29 colon carcinoma cells and in N-MYC-expressing NB-1691 neuroblastoma cells. The modified ODC promoter may, therefore, be useful in achieving tissue-specific expression of target proteins in tumor cells that overexpress c- or N-MYC.

Does cryotherapy affect refractive error? Results from treated versus control eyes in the cryotherapy for retinopathy of prematurity trial.

PURPOSE: To evaluate the effect of cryotherapy on refractive error status between ages 3 months and 10 years in children with birth weights of less than 1251 g in whom severe retinopathy of prematurity (ROP) developed in one or both eyes during the neonatal period. DESIGN: Randomized clinical trial. PARTICIPANTS: Two hundred ninety-one children in whom severe ROP developed during the neonatal period. INTERVENTION: Cryotherapy for ROP. MAIN OUTCOME MEASURES: Cycloplegic Refraction METHODS: The children underwent repeated follow-up eye examinations, including cycloplegic retinoscopy, between 3 months and 10 years after term due date. Refractive error data from all eyes that were randomized to cryotherapy were compared with data from all eyes that were randomized to serve as controls. Refractive error data were also compared for a subset of children who had both a treated and a control eye that could be refracted. RESULTS: At all ages, the proportion of treated eyes that were unable to be refracted because of retinal detachment, media opacity, or pupillary miosis was approximately half the proportion of the control eyes that were unable to be refracted. When data from all eyes that could be refracted were considered, the distribution of refractive errors between fewer than 8 diopters (D) of myopia and more than 8 D of hyperopia was similar for treated and control eyes at all ages. The proportion of eyes with 8 D or more of myopia was much higher in treated than in control eyes at all ages after 3 months. In the subset of children who had a treated eye and a control eye that could be refracted, distributions of refractive errors in treated versus control eyes were similar at most ages. CONCLUSIONS: In both treated and control eyes, there was an increase in the prevalence of high myopia between 3 and 12 months of age. Between 12 months and 10 years of age, there was little change in distribution of refractive error in treated or control eyes. The higher prevalence of myopia of 8 D or more in treated eyes, as compared with control eyes, may be the result of cryotherapy's preservation of retinal structure in eyes that, in the absence of cryotherapy, would have progressed to retinal detachment.