A randomized controlled trial of fresh frozen plasma for coagulopathy in Russell's viper (Daboia russelii) envenoming.

Essentials Russell's viper envenoming is a major health issue in South Asia and causes coagulopathy. We studied the effect of fresh frozen plasma and two antivenom doses on correcting coagulopathy. Fresh frozen plasma did not hasten recovery of coagulopathy. Low-dose antivenom did not worsen coagulopathy. SUMMARY: Background Russell's viper (Daboia russelii) envenoming is a major health issue in South Asia and causes venom-induced consumption coagulopathy (VICC). Objectives To investigate the effects of fresh frozen plasma (FFP) and two antivenom doses in correcting VICC. Methods We undertook an open-label randomized controlled trial in patients with VICC at two Sri Lankan hospitals. Patients with suspected Russell's viper bites and coagulopathy were randomly allocated (1 : 1) to high-dose antivenom (20 vials) or low-dose antivenom (10 vials) plus 4 U of FFP. The primary outcome was the proportion of patients with an International Normalized Ratio (INR) of < 2 at 6 h after antivenom administration. Secondary outcomes included anaphylaxis, major hemorrhage, death, and clotting factor recovery. Results From 214 eligible patients, 141 were randomized: 71 to high-dose antivenom, and 70 to low-dose antivenom/FFP; five had no post-antivenom blood tests. The groups were similar except for a delay of 1 h in antivenom administration for FFP patients. Six hours after antivenom administration, 23 of 69 (33%) patients allocated to high-dose antivenom had an INR of < 2, as compared with 28 of 67 (42%) allocated to low-dose antivenom/FFP (absolute difference 8%; 95% confidence interval - 8% to 25%). Fifteen patients allocated to FFP did not receive it. Severe anaphylaxis occurred equally frequently in each group. One patient given FFP developed transfusion-related acute lung injury. Three deaths occurred in low-dose antivenom/FFP patients, including one intracranial hemorrhage. There was no difference in recovery rates of INR or fibrinogen, but there was more rapid initial recovery of factor V and FX in FFP patients. Conclusion FFP after antivenom administration in patients with Russell's viper bites did not hasten recovery of coagulopathy. Low-dose antivenom/FFP did not worsen VICC, suggesting that low-dose antivenom is sufficient.

Circulating CD36+ microparticles are not altered by docosahexaenoic or eicosapentaenoic acid supplementation.

BACKGROUND AND AIMS: Circulating microparticles (MP) are the source of a plasma derived form of the scavenger receptor CD36, termed soluble (s)CD36, the levels of which correlate with markers of atherosclerosis and risk of cardiovascular disease. Long chain n-3 polyunsaturated fatty acids have cardioprotective effects that we have previously reported to be gender specific. The aim of this study was to determine if dietary docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA) supplementation affect circulating CD36 + MP levels, and if this occurs differentially in healthy men and women. METHODS AND RESULTS: Participants (43M, 51F) aged 39.6 ± 1.7 years received 4 weeks of daily supplementation with DHA rich (200 mg EPA; 1000 mg DHA), EPA rich (1000 mg EPA; 200 mg DHA), or placebo (sunola) oil in a double-blinded, randomised, placebo controlled trial. Plasma CD36 + MP were enumerated by flow cytometry and differences between genders and treatments were evaluated by Student's or paired t-test and one way ANOVA. Males and females had similar levels of CD36 + MP at baseline (mean = 1018 ± 325 vs 980 ± 318; p = 0.577) and these were not significantly changed after DHA (M, p = 0.571; F, p = 0.444) or EPA (M, p = 0.361; F, p = 0.901) supplementation. Likewise, the overall percent change in these levels were not different between supplemented cohorts compared to placebo when all participants were combined (% change in CD36 + MP: DHA = 5.7 ± 37.5, EPA = -3.4 ± 35.4, placebo = -11.5 ± 32.9; p = 0.158) or stratified by gender (M, DHA = -2.6 ± 30.6, EPA = -15.1 ± 20.1, placebo = -21.4 ± 28.7, p = 0.187; F, DHA = 11.7 ± 41.5, EPA = 6.8 ± 42.9, placebo = -2.8 ± 34.7, p = 0.552). CONCLUSION: The cardioprotective effects of DHA and EPA do not act through a CD36 + MP mechanism.

Hump-nosed pit viper (Hypnale hypnale) envenoming causes mild coagulopathy with incomplete clotting factor consumption.

CONTEXT: Limited information exists on the coagulopathy caused by hump-nosed pit viper (Hypnale hypnale) envenoming. OBJECTIVES: This study aimed to characterise the coagulopathy in hump-nosed pit viper bites by measuring laboratory clotting times and factor studies. MATERIALS AND METHODS: Cases of hump-nosed pit viper envenoming were included from a prospective cohort study of Sri Lankan snake-bite patients. Patient age, sex, snake identification, time of bite and clinical effects were recorded. Patients did not receive anti-venom because no specific anti-venom to hump-nosed vipers exists. All patients received supportive care and serial 20-min whole blood clotting tests (WBCT20). The prothrombin time (PT), international normalised ratio (INR), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, von Willebrand factor (vWF) antigen and D-Dimer concentrations were measured. The median of highest or lowest test result for each patient was reported with interquartile range (IQR). Results. There were 80 hump-nosed pit viper bites, median age was 37 years (IQR: 26-51 years) and 48 were male. The WBCT20 was positive in one patient. The median highest INR was 1.9 (1.5-2.2; Range: 1.3 to > 12) and median highest aPTT was 54 s (46-72 s; Range: 35-170 s). There was low fibrinogen [median: 1.3 g/L;1, -1.8 g/L; Range: < 0.2-2.9], low factor VIII levels [median: 23%; 16-37%] and low factor V levels [median: 43%; 23-74%]. D-Dimer concentrations [median: 3.4 mg/L; 2-7.4 mg/L] were slightly elevated. Factors II, VII and X and vWF antigen concentrations were normal. DISCUSSION AND CONCLUSIONS: Hump-nosed pit viper bites result in a mild coagulopathy which is usually not detected by a WBCT20. It is characterised by mild elevation of INR, low fibrinogen and Factors V and VIII which may be consistent with the venom containing a thrombin-like enzyme.

A randomized controlled trial of fresh frozen plasma for treating venom-induced consumption coagulopathy in cases of Australian snakebite (ASP-18).

BACKGROUND: Venom-induced consumption coagulopathy (VICC) is a major effect of snake envenoming. OBJECTIVES: To investigate whether fresh frozen plasma (FFP) given after antivenom resulted in more rapid correction of coagulation. PATIENTS/METHODS: This was a multicenter open-label randomized controlled trial in patients with VICC of FFP vs. no FFP within 4 h of antivenom administration. Patients (> 2 years) recruited to the Australian snakebite project with VICC (International Normalized Ratio [INR] > 3) were eligible. Patients were randomized 2 : 1 to receive FFP or no FFP. The primary outcome was the proportion with an INR of < 2 at 6 h after antivenom administration. Secondary outcomes included time from antivenom administration to discharge, adverse effects, major hemorrhage, and death. RESULTS: Of 70 eligible patients, 65 consented to be randomized: 41 to FFP, and 24 to no FFP. Six hours after antivenom administration, more patients randomized to FFP had an INR of < 2 (30/41 [73%] vs. 6/24 [25%]; absolute difference, 48%; 95% confidence interval 23-73%; P = 0.0002). The median time from antivenom administration to discharge was similar (34 h, range 14-230 h vs. 39 h, range 14-321 h; P = 0.44). Seven patients developed systemic hypersensitivity reactions after antivenom administration - two mild and one severe (FFP arm), and three mild and one severe (no FFP). One serious adverse event (intracranial hemorrhage and death) occurred in an FFP patient with pre-existing hypertension, who was hypertensive on admission, and developed a headache 6 h after FFP administration. Post hoc analysis showed that the median time from bite to FFP administration was significantly shorter for non-responders to FFP than for responders (4.7 h, interquartile range [IQR] 4.2-6.7 h vs. 7.3 h, IQR 6.1-8 h; P = 0.002). CONCLUSIONS: FFP administration after antivenom administration results in more rapid restoration of clotting function in most patients, but no decrease in discharge time. Early FFP administration (< 6-8 h) post-bite is less likely to be effective.

The origin of circulating CD36 in type 2 diabetes.

OBJECTIVE: Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36+MPs in persons with T2DM. DESIGN: An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI)=39.9±6.4 kg m(-2); age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI=23.6±1.8 kg m(-2) and 33.5±5.9 kg m(-2), respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235a, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations. RESULTS: CD36+MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235a+=35.8±14.6%); although this did not correlate with haemoglobin A(1c). By contrast, the main source of CD36+MPs in non-T2DM individuals was endothelial cells (CD105+=40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 µg ml(-1) and was positively correlated with CD36+MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36+MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively). CONCLUSIONS: Both the levels and cellular profile of CD36+MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.

Predicting 5-fluorouracil toxicity in colorectal cancer patients from peripheral blood cell telomere length: a multivariate analysis.

BACKGROUND: Identifying various pretreatment factors that predict chemotherapy-induced toxicity in colorectal cancer (CRC) patients undergoing treatment for their disease is crucial to optimising patient care. METHODS: Seventy-three patients received adjuvant 5-fluorouracil (5FU)/leucovorin using either the Mayo Clinic (n=42) or a weekly schedule (n=31) and evaluated for clinical toxicity. Pretreatment blood analysis included measures of plasma uracil and dihydrouracil, peripheral blood mononuclear cell (PBMNC) telomere length (TL), standard biochemistry and cell differential analysis. On the first day of treatment 5FU-pharmacokinetic variables of area under the curve, half life and clearance were also measured. These variables together with age and gender were used in univariate and multivariate analysis as predictors of clinical toxicity. RESULTS: For the Mayo schedule the primary toxicities were neutropenia (69%), mucositis (58%) and leukopenia (46%), with 70% of patients presenting with haematological toxicity ≥grade 1 (neutropenia and/or leukopenia). Multivariate analysis showed that haematological toxicity was predicted by short TL, high platelet lymphocyte ratio (PLR) and low neutrophil count (R(2)=0.38, P<0.0006), whereas mucositis was predicted by age, TL and PLR (R(2)=0.34, P<0.001). For the weekly schedule diarrhoea predominated (16%), with female gender as the only predictive factor. Although measures of uracil metabolism correlated well with 5FU metabolism (r=0.45-0.49), they did not indicate abnormal pyrimidine metabolism in this cohort and not surprisingly failed to predict for 5FU toxicity. CONCLUSION: Short TL of PBMNC and an increased PLR were strong predictors of mucositis and haematological toxicity in CRC patients undergoing 5FU treatment in the adjuvant setting.

Gender-specific inhibition of platelet aggregation following omega-3 fatty acid supplementation.

BACKGROUND AND AIMS: Increased platelet aggregation is a major risk factor for heart attacks, stroke and thrombosis. Long chain omega-3 polyunsaturated fatty acids (LCn-3PUFA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) reduce platelet aggregation; however studies in the published literature involving EPA and/or DHA supplementation have yielded equivocal results. Recent in vitro studies have demonstrated that inhibition of platelet aggregation by LCn-3PUFA is gender specific. We examined the acute effects of dietary supplementation with EPA or DHA rich oils on platelet aggregation in healthy male and females. METHODS AND RESULTS: A blinded placebo controlled trial involving 15 male and 15 female subjects. Platelet aggregation was measured at 0, 2, 5 and 24 h post-supplementation with a single dose of either a placebo or EPA or DHA rich oil capsules. The relationship between LCn-3PUFA and platelet activity at each time point was examined according to gender vs. treatment. EPA was significantly the most effective in reducing platelet aggregation in males at 2, 5 and 24 h post-supplementation (-11%, -10.6%, -20.5% respectively) whereas DHA was not effective relative to placebo. In contrast, in females, DHA significantly reduced platelet aggregation at 24 h (-13.7%) while EPA was not effective. An inverse relationship between testosterone levels and platelet aggregation following EPA supplementation was observed. CONCLUSION: Interactions between sex hormones and omega-3 fatty acids exist to differentially reduce platelet aggregation. For healthy individuals, males may benefit more from EPA supplementation while females are more responsive to DHA.

The Fat1 cadherin is overexpressed and an independent prognostic factor for survival in paired diagnosis-relapse samples of precursor B-cell acute lymphoblastic leukemia.

Improved survival of patients with acute lymphoblastic leukemia (ALL) has emerged from identifying new prognostic markers; however, 20% of children still suffer recurrence. Previously, the altered expression of Fat1 cadherin has been implicated in a number of solid tumors. In this report, in vitro analysis shows that Fat1 protein is expressed by a range of leukemia cell lines, but not by normal peripheral blood (PB) and bone marrow (BM) cells from healthy donors. In silico analysis of expression of array data from clinical leukemias found significant levels of Fat1 transcript in 11% of acute myeloid leukemia, 29% and 63% of ALL of B and T lineages, respectively, and little or no transcript present in normal PB or BM. Furthermore, in two independent studies of matched diagnosis-relapse of precursor B-cell (preB) ALL pediatric samples (n=32 and n=27), the level of Fat1 mRNA expression was prognostic at the time of diagnosis. High Fat1 mRNA expression was predictive of shorter relapse-free and overall survival, independent of other traditional prognostic markers, including white blood cell count, sex and age. The data presented demonstrate that Fat1 expression in preB-ALL has a role in the emergence of relapse and could provide a suitable therapeutic target in high-risk preB-ALL.

The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles.

BACKGROUND: CD36 is a widely expressed cell surface receptor that binds lipoproteins, and its function has been implicated in many complications of the metabolic syndrome. A cell-free form of CD36, soluble CD36 (sCD36), has been reported in human plasma, found to be elevated in obesity and diabetes, and claimed as a marker of insulin resistance. OBJECTIVE: To determine the nature of sCD36; in particular, whether sCD36 is truly soluble or, as hypothesized, is found as a component of circulating microparticles (MPs). METHODS: Lipoproteins were fractionated by density gradient centrifugation, and plasma MPs were isolated by ultracentrifugation, size exclusion, and immunoprecipitation with CD36 detected by immunoblotting. MPs from plasma and activated platelets were analyzed by multicolor flow cytometry, with a DyLight-488 anti-CD36 conjugate in combination with antibodies against different cellular markers. RESULTS: Cell-free plasma CD36 was not observed associated with lipoproteins and was not a proteolytic fragment; rather, it was associated with the plasma MP fraction, suggesting that sCD36 in the plasma of normal subjects is a product of circulating MPs. Cytometric and immunoblotting analyses of plasma from normal donors showed that these MPs were derived mainly from platelets. Analysis of in vitro activated platelets also showed that CD36 to be secreted in the form of MPs. CONCLUSIONS: sCD36 is not a proteolytic product, but rather is associated with a specific subset of circulating MPs that can readily be analysed. This finding will enable more specific investigations into the cellular source of the increased levels of plasma CD36 found in subjects with diabetes.

Association of thymidylate synthase enhancer region polymorphisms with thymidylate synthase activity in vivo.

Two known polymorphisms in the 5' enhancer region (ER) of the thymidylate synthase (TS) gene, a variable number of tandem repeats of a 28 bp sequence (2R/3R) and a further G>C single nucleotide substitution within the repeats, result in genotypes with 0-5 functional upstream stimulatory factor (USF) E-box consensus elements. However, the relationship between these polymorphisms, regulation of TS expression and patient response to fluoropyrimidine treatment has been inconsistent. In this study, seven possible TSER allele configurations showed similar patterns of luciferase gene expression regardless of cell type or USF-1 content, with no significant difference in promoter activity between the wild-type 2RGC and 3RGGC (1.40±0.37 vs 1.43±0.32, P=0.90), whereas the minor alleles, 2RCC and 3RGCC, were significantly reduced (0.84±0.17, P=0.01) and increased (3.19±0.72, P=0.001) respectively. Patient plasma levels of 2'-deoxyuridine, a surrogate marker of TS activity, were significantly different between genotypes (P<0.001) and inversely related to luciferase activity (P=0.02) but not to the absolute number of functional repeated elements (P=0.16), suggesting that the position, rather than the number of functional USF E-box repeats in the TSER, is responsible for determining gene expression in vitro and TS activity in vivo.

Factor deficiencies in venom-induced consumption coagulopathy resulting from Australian elapid envenomation: Australian Snakebite Project (ASP-10).

BACKGROUND: Limited information exists on the dynamics of hemostasis in patients with venom-induced consumption coagulopathy (VICC) from snake envenomation. OBJECTIVE: The aim of the present study was to investigate specific factor deficiencies and their time course in Australasian elapid envenomation. METHODS: We measured coagulation parameters and factor concentrations in patients recruited to the Australian Snakebite Project, an observational cohort study. There were 112 patients with complete VICC, defined as an international normalized ratio (INR) > 3, and 18 with partial VICC. Serial citrated plasma samples were collected from 0.5 to 60 h post-bite. INR, activated partial thromboplastin time (aPTT), coagulation factors (F)I, II, V, VII, VIII, IX, X, von Willebrand factor antigen (VWF:Ag) and D-dimer concentrations were measured. RESULTS: Complete VICC was characterized by near/total depletion of fibrinogen, FV and FVIII, with an INR and aPTT that exceeded the upper limits of detection, within 2 h of snakebite. Prothrombin levels never fell below 60% of normal, suggesting that the toxins were rapidly eliminated or inactivated and re-synthesis of clotting factors occurred irrespective of antivenom. Partial VICC caused limited depletion of fibrinogen and FV, and almost complete consumption of FVIII. Onset of VICC was more rapid with brown snake (Pseudonaja spp.) venom, which contains a group C prothrombin activator toxin, compared with the tiger snake group, which contains a group D prothrombin activator toxin and requires human FVa formation. Resolution of VICC occurred within 24-36 h irrespective of snake type. CONCLUSIONS: These results suggest that Australasian elapid prothrombin activators have a potent but short duration of action. Antivenom is unlikely to be administered in time to prevent VICC.

Telomere length predicts neutrophil recovery in the absence of G-CSF after autologous peripheral blood stem cell transplantation.

SUMMARY: Haemopoietic regeneration after autologous peripheral blood progenitor cell (PBPC) transplantation can be delayed in some patients despite adequate infusion of CD34(+) cells. This suggests variability in the proliferation potential of the implanted cells, a capacity that may be predicted by their telomere length. To test this theory, telomere length was measured on stored apheresis samples from 36 patients aged 46.6+/-11.1 years, who had undergone successful autologous PBPC transplantation with a median of 5.6 x 10(6)/kg (1.3 x 10(6)-36.1 x 10(6)/kg) CD34(+) cells. The mean PBPC telomere length for the cohort was 9.4+/-2.3 kbp. For patients who did not receive G-CSF post transplantation (n=7), days to absolute neutrophil recovery (ANC), >/=0.1, 0.5 and 1.0 x 10(9) cells/l, were significantly inversely correlated with telomere length of the infused PBPC (r=-0.88, -0.81, -0.77, respectively; P<0.05,). However, no correlation was found for patients who received G-CSF from day 1 post transplantation (n=20). These data suggest that for transplantation with sufficient CD34(+) cells, neutrophil recovery is less efficient in patients receiving infusions of cells with short telomeres, but this deficiency can be corrected with adequate post transplantation administration of G-CSF. Bone Marrow Transplantation (2004) 34, 439-445. doi:10.1038/sj.bmt.1704607 Published online 19 July 2004

TRAIL-induced eradication of primary tumour cells from multiple myeloma patient bone marrows is not related to TRAIL receptor expression or prior chemotherapy.

TNF-related apoptosis-inducing ligand (TRAIL) shares significant homology with CD95 (Fas) ligand and has the ability to induce apoptosis in sensitive cells through a caspase-mediated pathway. We have evaluated the activity of purified human recombinant soluble TRAIL (S-TRAIL, comprising residues 114-281; Biomol, Plymouth Meeting, PA, USA) and a leucine zipper construct of TRAIL (LZ-TRAIL; Immunex, Seattle WA, USA) against myeloma cell lines NCI H929, U266, RPMI 8226, the FasL-sensitive Jurkat T cell ALL line, the lymphoblastoid cell line MC/CAR and primary tumour cells from 16 myeloma patients. Furthermore, we examined the relationship between TRAIL-induced apoptosis and TRAIL receptor expression utilising RT-PCR and flow cytometry. Two of three myeloma cell lines and Jurkat were TRAIL sensitive whereas MC/CAR was relatively resistant. Five of 16 (31%) primary tumours demonstrated > or =20% reduction in myeloma cells following TRAIL incubation (20-59%). This did not correlate with prior therapy. Four cell lines (two sensitive) and five primary tumours (two sensitive) demonstrated mRNA expression of the intra-cellular death domain containing TRAIL-R1. Variable expression of the two decoy (TRAIL-R3 and R4) and soluble (osteoprotegerin) receptors was seen and this did not correlate with TRAIL resistance. We conclude that myeloma cell expression of death effector receptors for TRAIL is insufficient to confer sensitivity to TRAIL-induced apoptosis but that in a significant minority of patients, irrespective of prior therapy, tumour cells are sensitive to TRAIL. The further investigation of TRAIL as an adjunct to presently available therapies for myeloma is justified.

Tumour kinetics in multiple myeloma before, during, and after treatment.

Tumour progression was monitored in seven multiple myeloma (MM) patients undergoing a novel oral chemotherapy regimen (cyclophosphamide, idarubicin and dexamethasone; CID) followed by early autologous stem cell transplantation (ASCT). Allele-specific oligonucleotide PCR (ASO-PCR) was used to semi-quantitate the number of tumour cells within the peripheral blood (PB) and PB progenitor cell (PBPC) harvests and compared with paraprotein levels and morphological bone marrow (BM) assessments. Tumour cells were detected in the PB of all patients at diagnosis, but decreased in response to CID therapy. All but two of the 22 PBPC collections contained MM cells, the levels of which were statistically correlated with overall clinical response to therapy, but not with individual BM or PB tumour loads prior to mobilisation. We also found no correlation between the day of leucapheresis collection and the number of contaminating MM cells, CD34+ cells or MM cells per CD34+ cell. Regardless of tumour contamination levels in the PBPC collections, the majority of patients demonstrated post-ASCT clearing of circulating MM cells. This study suggests that levels of circulating MM cells may be the best indication of patient response to treatment and argues against the theory of differential mobilisation of tumour cells and CD34+ cells in response to cytokine treatment.

Deciphering the apoptotic pathway: all roads lead to death.

Research into apoptosis is proceeding at such a fast and ferocious pace that anyone who is not completely engrossed in the field has difficulty keeping track of the constant stream of newly identified proteins involved in the process. Apart from being an enticing concept, the process of cell suicide is an important function with wide-reaching implications. Virologists, biologists, immunologists, physiologists and oncologists alike have had to incorporate this phenomenon into their disciplines. The purpose of this article is to provide a solid background on which to further review recent advances in this exciting field. The Bcl-2 and caspase family homologues are discussed in detail and various models are proposed to explain how they function to regulate and execute the death programme. Finally, the importance of programmed cell death with respect to immune function is explored, emphasizing the targets of viral inhibitors of apoptosis.

Formation of spheroid structures in a human colon carcinoma cell line involves a complex series of intercellular rearrangements.

The structural remodelling of tissues that occurs in vivo during animal morphogenesis can often prove difficult to study. Here we investigate the organizational processes of the LIM 1863 colon carcinoma cell line as it transforms from a single-cell stage into multicellular spherical structures called 'organoids'. The organoids can be dissociated into a viable single-cell suspension when cultured in calcium-depleted medium, and then induced to reform the organoid structure by the readdition of calcium. Previous studies have shown that initial cell attachment under these conditions is characterized by a novel mechanism of cell engulfment termed 'clutching'. This investigation reveals the subsequent appearance of junctional complexes between groups of 'clutched' cells prior to lumen formation, and the ultimate 'declutching' of entrapped cells as a means of cell rearrangement. Intact actin filaments but not microtubules were required for the initial clutching events, while inhibition of microtubule polymerization resulted in aberrant apical protein polarization, but did not affect the development of a luminal space within the spheroids. Single cells exhibited pools of intracellular microvilli contained in vacuolar apical compartments, which were resistant to the effects of cytoskeleton-disrupting drugs. However, these structures did not seem to be responsible for the swift development of the luminal surface observed in these cells. Two other cell lines, MDCK and DU 4475, were found to exhibit similar clutching conformations when induced to form three-dimensional structures, suggesting that this may be a widespread mechanism of cell rearrangement that reflects the process of organ morphogenesis in vivo.

Biochemical isolation of a membrane microdomain from resting platelets highly enriched in the plasma membrane glycoprotein CD36.

Here we describe the isolation and characterization of a Triton X-100-insoluble fraction isolated from lysates of platelets by flotation in sucrose gradients. Transmission electron microscopy of the insoluble material revealed a heterogeneous population of vesicles ranging in size from 20 to 1000 nm, and Western blot analyses of platelet lysates for the caveolae structural coat protein, caveolin/VIP21, were negative. Biochemical characterization of the Triton X-100-insoluble fraction showed it to be cholesterol-rich, greatly and specifically enriched in the plasma membrane glycoprotein CD36, and also to contain Src and the Src-related kinase, Lyn. CD36 within this fraction is shown to be palmitoylated, but the fraction itself is not generally enriched in palmitoylated platelet proteins. These results suggest that this fraction represents caveolin-negative, CD36-rich microdomains in the resting platelet membrane. CD36 can form associations with certain Src-related kinases and can signal to activate platelets. These results suggest the possibility that such microdomains are implicated in platelet activation.

Involvement of integrins in cell survival.

Apoptosis is a regulated process of cell death by which cells actively participate in their own destruction. In multicellular organisms, the balance between cell proliferation and apoptosis provides homeostatic control, and a regulatory failure of either event can contribute to oncogenesis. The extracellular matrix (ECM) is known to play a regulatory role in cellular growth and differentiation, but only more recently has it been recognized as a regulator of apoptosis. In these processes the major transmitters of ECM-derived signals to the cell are members of the integrin family, although the mechanical process of cell spreading also plays a role. Both in vivo and in vitro the loss of adhesion to specific components of the ECM can lead to cell death, and such apoptosis can be induced experimentally by blocking integrin binding. Heterotypic and homotypic cell-cell adhesion can also protect from adhesion-dependent apoptosis and there is evidence to suggest that this too in integrin mediated. In addition, some integrin mediated signaling appears to promote apoptosis. The downstream mechanisms of integrin signaling causing cell death have not been greatly explored, but there is evidence from two different systems that the induction of ICE transcription and nuclear translocation of p53 are candidate processes. Alterations in integrin expression or signaling therefore are likely to contribute to tumor development by enabling escape from apoptosis. Also, the recognition of the importance of cell-cell adhesion in tumor cell survival offers the potential of developing improved drug regimes for the treatment of malignancy.