Local and global aspects of biological motion perception in children born at very low birth weight.

Biological motion perception can be assessed using a variety of tasks. In the present study, 8- to 11-year-old children born prematurely at very low birth weight (<1500 g) and matched, full-term controls completed tasks that required the extraction of local motion cues, the ability to perceptually group these cues to extract information about body structure, and the ability to carry out higher order processes required for action recognition and person identification. Preterm children exhibited difficulties in all 4 aspects of biological motion perception. However, intercorrelations between test scores were weak in both full-term and preterm children--a finding that supports the view that these processes are relatively independent. Preterm children also displayed more autistic-like traits than full-term peers. In preterm (but not full-term) children, these traits were negatively correlated with performance in the task requiring structure-from-motion processing, r(30) = -.36, p < .05), but positively correlated with the ability to extract identity, r(30) = .45, p < .05). These findings extend previous reports of vulnerability in systems involved in processing dynamic cues in preterm children and suggest that a core deficit in social perception/cognition may contribute to the development of the social and behavioral difficulties even in members of this population who are functioning within the normal range intellectually. The results could inform the development of screening, diagnostic, and intervention tools.

Downregulation of Bcl-2 sensitises interferon-resistant renal cancer cells to Fas.

Interferon alpha (IFNalpha) is used to treat patients with advanced renal cell carcinoma (RCC) despite limited clinical benefit. IFNalpha can induce Fas receptor-mediated apoptosis by direct activation of pro-caspase-8 followed by activation of caspase-3. Alternative, indirect activation of caspase-3 via mitochondrial release of cytochrome c can occur and may explain the rescue from Fas-activated cell death by the antiapoptotic members of the Bcl-2 family. In this study, we examined G3139, a novel antisense compound targeting Bcl-2, in combination with IFNalpha. Human RCC lines (SK-RC-44 and SK-RC-07) were treated with IFNalpha, G3139 or a combination of the two. Fas-mediated cytotoxicity was induced by anti-Fas mAb, CH11. An analysis of Bcl-2, Fas and the cleavage of PARP was performed. IFNalpha induced Fas and Bcl-2 in SK-RC-44 and SK-RC-07. IFNalpha sensitised SK-RC-44 to anti-Fas and induced PARP cleavage confirming that IFNalpha has a cytotoxic effect on RCC lines by induction of the Fas antigen. Cytotoxicity was not evident in SK-RC-07 cells treated with IFNalpha. G3139 induced a specific downregulation of Bcl-2 in SK-RC-07 cells, which were then sensitised to anti-Fas after treatment with IFNalpha. Taken together, these results suggest that Fas-dependent pathways as well as alternative pathways, which can be inhibited by Bcl-2, exist in renal cell carcinoma. G3139 in combination with IFNalpha is a potential therapy in patients with metastatic renal cell carcinoma.

The effect of antisense Bcl-2 oligonucleotides on Bcl-2 protein expression and apoptosis in human bladder transitional cell carcinoma.

PURPOSE: Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model. RESULTS: Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death. CONCLUSIONS: Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.

Antisense Bcl-2 oligonucleotide uptake in human transitional cell carcinoma.

OBJECTIVES: Antisense oligonucleotides (AO) downregulate Bcl-2 protein expression in various tumours if good target cell uptake is achieved. In this study, uptake of FITC labelled AO (FITC-AO) directed at Bcl-2 was examined in: (1) the RT4 bladder tumour cell line; (2) normal pig urothelium, and (3) human superficial bladder tumours. METHODS: In the RT4 cell line, uptake of FITC-AO, FITC-scrambled and FITC-sense oligonucleotides were quantified by flow cytometry at 4-hour intervals over 24 h. Uptake of FITC-AO was assessed in normal pig urothelium by flow cytometry after FITC-AO was infused for 1 h. Uptake of FITC AO was assessed in samples from 14 human superficial bladder tumours which were maintained in an ex vivo model. In samples from 6 tumours, uptake at 4 h was assessed using fluorescence microscopy. In samples from 8 separate tumours uptake every 4 h within the first 24-hour incubation period was assessed by flow cytometry. RESULTS: In the RT4 cell line the FITC-AO, FITC-scrambled and FITC-sense oligonucleotide uptake was similar. Disaggregated cells from the normal urothelium of the 3 pigs exhibited 33, 46 and 51% of cells staining positively for FITC-AO as determined by flow cytometry. All 6 tumour samples had detectable intracellular FITC-AO by fluorescence microscopy at 4 h. In the 8 tumours examined over the 24-hour incubation period, there was a range of percentages of positively staining cells. However, most tumours had a monotonic increase in intracellular fluorescence intensity that plateaued 16 h post-infusion. CONCLUSION: Antisense Bcl-2 oligonucleotides were readily taken up by superficial bladder cancer cells but the heterogeneous uptake in tumour samples needs to be considered when assessing the bioavailability of these drugs.

Inhibition of amyloid fiber assembly by both BiP and its target peptide.

Immunoglobulin light chain (LC) normally is a soluble, secreted protein, but some LC assemble into ordered fibrils whose deposition in tissues results in amyloidosis and organ failure. Here we reconstitute fibril formation in vitro and show that preformed fibrils can nucleate polymerization of soluble LC. This prion-like behavior has important physiological implications, since somatic mutations generate multiple related LC sequences. Furthermore, we demonstrate that fibril formation in vitro and aggregation of whole LC within cells are inhibited by BiP and by a synthetic peptide that is identical to a major LC binding site for BiP. We propose that LC form fibrils via an interprotein loop swap and that the underlying conformational change should be amenable to drug therapy.

Induction of apoptosis by mitomycin-C in an ex vivo model of bladder cancer.

OBJECTIVE: To examine mitomycin-C (MMC)-induced apoptosis in an ex vivo model of superficial TCC, and relate it to the in vivo response to chemotherapy. Materials and methods Dose- and time-response curves were constructed to determine the optimal conditions for the induction of apoptosis by MMC in an ex vivo model of superficial bladder cancer. Subsequently, 41 individual tumours were exposed to MMC in the model and the effects assessed by measuring of apoptosis before and after chemotherapy. The relationships between tumour grade and stage and the intrinsic and induced apoptotic counts were determined. In tandem, in a clinical study, the relationship between in vivo response of a marker tumour to MMC and the ex vivo induction of apoptosis was determined. RESULTS: In the ex vivo model, apoptosis was induced at a MMC concentration of 0.5 mg/mL after an incubation time of 8 h. In 41 tumours the intrinsic apoptotic index (AI) was higher with increased grade and stage of tumour (P = 0.048). There was no correlation between the intrinsic AI and the AI after treatment with MMC (induced AI). In 21 tumours (51%) the induced AI did not increase above a predetermined response threshold and these tumours were considered resistant to MMC. Resistance to MMC was related to tumour grade (P = 0.037) with a trend for G3 pT1 tumours to be resistant to the therapy. There was a significant association between ex vivo sensitivity and in vivo marker tumour response (P = 0.02). CONCLUSIONS: Apoptosis is differentially induced in an ex vivo incubation model of superficial TCC by MMC and evidence suggests that this response matches that seen in vivo. The measurement of apoptosis before therapy does not predict the apoptotic response of a tumour to chemotherapy. The ability to undergo apoptosis correlates with clinical outcome.

The effects of l-arginine on crypt cell hyperproliferation in colorectal cancer.

BACKGROUND: Colonic crypt cell hyperproliferation characterizes malignant and premalignant conditions of the colon and may be modified by dietary manipulation. This study compared the effect of dietary arginine supplementation on colonic crypt cell proliferation during the initiation and promotion stages of colorectal carcinogenesis. MATERIALS AND METHODS: One hundred and twenty male Wistar rats were divided into 5 groups of 24 animals each. Groups D, DA, FA, and LA received subcutaneous injections of 1, 2-dimethylhydrazine for 20 weeks. Group D received no arginine supplement. l-arginine was given as a 1% solution instead of drinking water to Group DA for 22 weeks, to Group FA for the first 10 weeks, and to Group LA for the last 12 weeks. EDTA animals were given subcutaneous injections of EDTA for 20 weeks. Colonic crypt cell proliferation was assessed in 6 animals from each of the five groups and in 6 normal rats not given DMH or EDTA. RESULTS: The BrdUrd-labeling index and proliferative zone were significantly decreased in all arginine groups (DA, FA, LA). The greatest reduction was evident in Group FA in which tumor incidence and tumor size were also significantly lowered. CONCLUSIONS: When given during the initiation phase of carcinogenesis l-arginine significantly reduced colorectal tumor production and crypt cell hyperproliferation.

DNA denaturation sensitivity may invalidate bromodeoxyuridine--DNA flow cytometric analysis of potential doubling times in colorectal tumours.

BACKGROUND: Flow cytometric analysis of 5-bromo-2'-deoxyuridine (BrdU)-substituted DNA has been used to calculate tumour cell proliferation rate. In this methodology, DNA denaturation, commonly by hydrochloric acid, is essential to expose incorporated BrdU for quantification with monoclonal antibodies. This study was designed to establish the validity of this technique by examining the change in flow cytometric DNA profiles introduced by DNA denaturation procedures. METHODS: Four experiments were performed using suspensions of nuclei derived from human colorectal tumours exposed in vivo to 150 mg/m2 BrdU 8-11 h before sampling. RESULTS: After denaturation with hydrochloric acid 2 mol/l a significant decrease was observed in the DNA aneuploid G1 population (P < 0.001) with a concurrent increase in the DNA aneuploid S phase fraction (P < 0.05). These changes were independent of the washing-centrifugation step and were maximal at different hydrochloric acid concentrations for different tumours. CONCLUSION: Hydrochloric acid denaturation introduces a tumour-specific non-linear variation in the analysis of BrdU.

Validation of a rapid method to quantify apoptosis in superficial bladder cancer.

OBJECTIVES: To derive and validate a rapid method for calculating apoptotic indices in superficial transitional cell carcinoma (TCC) as a measure of chemosensitivity to mitomycin. MATERIALS AND METHODS: Apoptotic cells, identified by light microscopy in 20 superficial TCC specimens, were expressed as an index of the total tumour cell population within defined fields. For a given field, the total cell population was estimated by: (i) an exhaustive count of the total number of cells in the field and (ii) an abbreviated method in which the number of cells in a subfield was multiplied to provide an estimate of the total field number. Field and specimen estimates were compared using agreement statistics and the intra- and inter-observer reproducibility of apoptotic indices calculated. RESULTS: Cellularity and apoptotic indices obtained using method (ii) were correlated significantly with the true cell counts (P < 0.001). Agreement statistics showed that only 9.4% of counts fell outside two standard deviations (SD) from the mean in field analysis, and only 10% of counts fell outside 2 SD from the mean in specimen analysis. There was a fivefold variation in tumour cell counts among individual fields. CONCLUSIONS: The reported variation in cellularity among fields shows that the calculation of apoptosis must use the total cell population as the reference. The limits of agreement for the estimated and true cell counts are small enough to be confident that the shorter method to estimate cellularity can be used in place of counting all cells.

Colorectal cell kinetics.

The assessment of cell proliferation in colorectal tissue may provide information with both prognostic and therapeutic implications. A variety of methods are available, including flow cytometric estimations of S phase fraction, immunohistochemical and autoradiographic visualization of exogenous and endogenous proliferation proteins, and morphological and stathmokinetic techniques. There is some correlation between Dukes stage and proliferation state features, and there is increased proliferative activity throughout the adenoma-carcinoma sequence. Data on cell proliferation rates are difficult to obtain. When correctly applied, the metaphase arrest technique remains the 'gold standard' of measuring proliferation, but its usefulness in clinical practice is limited. Recent studies have employed dual measurement flow cytometry and double labelling techniques to produce rate data.

Three-dimensional computerised analysis of epithelial cell proliferation in the gastrointestinal tract.

This study describes a new technique for the visualisation and quantitation of glandular epithelial cell proliferation in gastrointestinal mucosa using computerised three-dimensional reconstruction. The tissue used in this study was colorectal biopsy tissue infiltrated in vitro with bromodeoxyuridine (BrdU), although the method could be applied to any gastrointestinal site labelled with any specific marker for cell proliferation. The method is as follows. Five-micron-thick serial sections (> 100) were cut from colorectal biopsies infiltrated in vitro with BrdU. After labelling all the sections for BrdU-positive cells using standard immunohistochemistry, colorectal glands were identified which were completely sectioned within the series. Each microscopic image of the sectioned gland was orientated, digitised and stored using a Kontron image analyser. On each of the stored images, the crypt profile, the positive cells and the negative cells were interactively marked and digitally stored. Using three-dimensional (3-D) reconstruction software, the outer surface of the crypt, the total positive and the total negative fractions could be viewed in three dimensions. The total BrdU-positive cell number could be automatically calculated for the complete crypt or, alternatively, compartmental analysis of the labelling pattern within the crypt could be obtained. This represents a powerful technique: it does not require orientation, it can be carried out on complex glandular structures and is not affected by the biases involved in measuring labelling indices from single tissue sections.