Clinical relevance of flow cytometric immunophenotyping of the cerebrospinal fluid in patients with diffuse large B-cell lymphoma.

BACKGROUND: Central nervous system (CNS) relapse is an uncommon but dramatic complication of diffuse large B-cell lymphoma (DLBCL). Several studies have demonstrated the superiority of cerebrospinal fluid (CSF) flow cytometry (FCM), as compared with conventional cytology (CC), in detecting occult leptomeningeal disease. The clinical relevance of a positive FCM still has to be clarified. PATIENTS AND METHODS: We analyzed CSF from 114 DLBCL patients at diagnosis (n = 95) or at relapse (n = 19) by FCM and CC. Most patients received meningeal prophylaxis. FCM results did not influence treatment strategies. RESULTS: Fourteen samples were FCM+, versus one CC+ (also FCM+). Within all patients without neurological symptoms (n = 101), four (4%) relapsed in the CNS, with a median time to relapse of 5.2 months. Only one-fourth (25%) was FCM+ before relapse. More than one extranodal disease site and elevated lactate dehydrogenase levels were associated with an increased risk of CNS relapse. CONCLUSIONS: FCM gives far more positive results than CC. However, a positive FCM result did not translate into a significant increase in CNS relapse rate in this histologically uniform population receiving CNS prophylaxis.

Beat wave interferometry for measuring relative motion.

A new approach to detecting relative motion is proposed based on measuring the beat frequency. A formula is derived for the beat frequency as a function of the speed of translation. A principal sketch of an experimental setup implementing the new idea is proposed, and the effects of possible small differences in the frequencies and phases of the sources are discussed.

A model for glioma cell migration on collagen and astrocytes.

We present a model for the migration of glioma cells on substrates of collagen and astrocytes. The model is based on a cellular automaton where the various dynamical effects are introduced through adequate evolution rules. Using our model, we investigate the role of homotype and heterotype gap junction communication and show that it is possible to reproduce the corresponding experimental migration patterns. In particular, we confirm the experimental findings that inhibition of homotype gap junctions favours migration while heterotype inhibition hinders it. Moreover, the effect of heterotype gap junction inhibition dominates that of homotype inhibition.

A cellular automaton model for the migration of glioma cells.

We present a study of in vitro cell migration in two dimensions as a first step towards understanding the mechanisms governing the motility of glioma cells. Our study is based on a cellular automaton model which aims at reproducing the kinetics of a lump of glioma cells deposited on a substrate of collagen. The dynamical effects of cell attraction and motion inertia are introduced through adequate automaton rules. We compare the density profiles given by the model to those obtained experimentally. The result of the best fit indicates a substantial cell-cell attraction due to cell-cell communication through gap junctions (or chemotaxis) and negligible inertia effects during migration. Tracking of individual migrating cells indicates highly convoluted cell trajectories.

Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma.

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

Primary brain lymphoma cell turnover differs in patients with and without AIDS: relationships to bcl-2 expression and host cell reaction.

Primary central nervous system lymphomas (PCNSLs) are more resistant to radiotherapy and chemotherapy in AIDS (A-PCNSLs) than in non-AIDS patients (NA-PCNSLs). We investigated 23 A-PCNSLs and 24 NA-PCNSLs. Lymphoma cell kinetics (i.e. proliferation [mitotic index, MIB-1 and PCNA labeling indices], apoptosis and turnover) were determined and compared with bcl-2 and LMP-1 expression, and to the percentage of tumor-infiltrating T-lymphocytes (T-TILs) and macrophages. A-PCNSLs showed lower proliferation (p < 0.005), less apoptosis (p < 0.0001) and slower cell-turnover (p < 0.0001) than NA-PCNSLs. LMP-1 was detected in 90% of A-PCNSLs and 5% of NA-PCNSLs, a finding correlating positively with bcl-2 expression (p < 0.0007). In contrast, T-TIL counts and CD4/CD8 T-TIL ratios were similar in A-PCNSLs and NA-PCNSLs. T-TIL counts correlated negatively with proliferation indices (from p < 0.05 to p < 0.0005) in NA-PCNSLs, but not in A-PCNSLs. Macrophage counts correlated positively with apoptosis in both groups. We concluded the following: (i) A-PCNSLs are characterized by accumulation of slow-cycling, long-lived cells that might be protected from apoptosis by LMP-1 induced bcl-2 expression, and independently from the host response; (ii) NA-PCNSLs are characterized by a faster cell turnover associated with an insufficient antiproliferative host response; and (iii) A-PCNSLs and NA-PCNSLs constitute 2 entities with distinctive morphology and different kinetic profiles that could account for different responses to therapy.

Two methods for rapid assembly and oligomerization of synthetic genes: construction of human calcitonin-encoding sequences.

Two fast, simple and reliable methods for construction of synthetic genes are described. The first represents a one-vial method for assembly and cloning of monomeric genes, and the second allows a simultaneous assembly, oligomerization and cloning of synthetic genes. Both methods consist of ligation of the synthetic oligodeoxyribonucleotides (oligos) building up the gene directly into the cloning vector, followed by screening of the recombinant clones for the presence of specific gene constructs. The average length of the oligomeric genes thus constructed is directly proportional to the molar ratio between the vector DNA and the synthetic oligos used. Both methods are illustrated by the construction of a series of human calcitonin-encoding genes.

Matrix metalloproteinase-9 is increased and correlates with severity in Guillain-Barré syndrome.

OBJECTIVE: To study the expression and activity of matrix metalloproteinases (MMPs) MMP-2 (72-kd type IV collagenase, gelatinase A), MMP-3 (58-kd stromelysin-1), and MMP-9 (92-kd type IV collagenase, gelatinase B) and tissue inhibitors of MPs (TIMP) in patients with Guillain-Barre syndrome (GBS). BACKGROUND: MMPs are able to proteolysis of basement membranes and other matrix components, promoting transmigration of inflammatory cells from circulation to nerve tissue. METHODS: Twenty-five patients with GBS were analyzed according to the phase of the disease, i.e., progression, plateau, early recovery, and late recovery. Determinations of MMP-2, MMP-3, MMP-9, and TIMP-1 were performed using ELISA, zymography, and immunocytochemistry in circulation or peripheral nerve. RESULTS: MMP-9 plasma levels were increased in 67% of patients on admission and decreased from progression to late recovery (p < 0.002). During the course of GBS, MMP-9 was progressively balanced by its inhibitor TIMP-1, as assessed by the MMP-9/TIMP-1 ratio. MMP-9 and TIMP-1 plasma levels and the MMP-9/TIMP-1 ratio correlated positively with disability. MMP-2 expression was similar to controls. MMP-3 activity was not detected, and plasma levels were not different from those in controls. Positive MMP-9 immunolabeling was 51 +/- 11% of circulating lymphocytes. It was observed in some endothelial cells and mononuclear cells adherent to the endothelium and close to myelinated fibers. CONCLUSIONS: Circulating matrix metalloproteinases (MMP-9) correlates with disease severity in Guillain-Barré syndrome (GBS). MMP-9 likely represents an important molecule in the pathogenesis of GBS and therefore could represent an interesting therapeutic target.

Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation.

Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT-PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4- and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention.

Heat conduction paradox involving second-sound propagation in moving media.

In this Letter, we revisit the Maxwell-Cattaneo law of finite-speed heat conduction. We point out that the usual form of this law, which involves a partial time derivative, leads to a paradoxical result if the body is in motion. We then show that by using the material derivative of the thermal flux, in lieu of the local one, the paradox is completely resolved. Specifically, that using the material derivative yields a constitutive relation that is Galilean invariant. Finally, we show that under this invariant reformulation, the system of governing equations, while still hyperbolic, cannot be reduced to a single transport equation in the multidimensional case.

Recurrent central neurocytoma with marked increase in MIB-1 labelling index.

Central neurocytomas (CNs) are characterized by an indolent growth and a low recurrence rate. Cell proliferation has been rarely studied in recurrent CNs despite the growing awareness of its prognostic value. We present a CN that recurred rapidly after a prolonged relapse-free interval showing a 4-fold increase of MIB-1 labelling index.

A conditional high-copy-number plasmid derivative of pBR322.

A spontaneous high-copy-number plasmid derivative of plasmid pBR322 was isolated. This plasmid bears two point mutations adjacent to the unpaired region in the stem of loop II of RNA I and expresses its high-copy-number phenotype only when the harboring cells grow on solid support (L-agar).

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptor flt-1 in microcystic meningiomas.

We investigated the immunohistochemical expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and of its endothelial cell receptor flt-1 in relationship to microcyst formation in meningiomas. Expression of VPF/VEGF was studied in 60 meningiomas (6 microcystic, 38 partially microcystic and 16 with no microcystic areas) and 30 meningiomas from these three subgroups were evaluated for flt-1 expression. VPF/VEGF immunoreactivity was mainly observed in vessel endothelium. Positive vessels were present in 75% (33/44) of meningiomas with any amount of microcystic pattern and in 38% (6/16) of the solid meningiomas (P < 0.02). Densities and percentages of both VPF/VEGF-positive and flt-1-positive vessels were higher in meningiomas with microcystic areas than in solid meningiomas (P /= 0.75, P < 0.0001). A strong positive correlation between VPF/VEGF-positive vessel density and proportion of microcystic pattern in all 60 specimens was found (r = 0.75, P < 0. 0001). We conclude that accumulation of flt-1-bound VPF/VEGF on endothelial cells of meningiomas is associated with microcyst formation that leads to the histologic appearance of microcystic meningiomas.

In vitro evaluation of human muscle satellite cell migration prior to fusion into myotubes.

We developed a short-time assay to evaluate muscle satellite cell migration, based on the fact that during myogenic differentiation, myoblasts migrate preferentially towards high cellular density areas where myotubes would form. This assay consists of a computer-assisted count of cells within a randomly chosen field, performed every hour for eight hours, and compared with the cell number at the start time of the experiment. Nine primary myoblast cultures were tested in triplicate. The method relies on several requisites. (1) Negligible cell proliferation: cell division was nearly absent in 8 h experiments. (2) Directional cell movement: a major flow of cells, either entering or exiting the fields, was constantly observed. 'Counter-flows', detected by visual counting, involved minor percentages of cells. (3) Constant migration rate: a linear increase in cell count variations over 8 h and a very high degree of intra-assay homogeneity were observed. Individual primary cell culture characteristics (depending on characteristics of the different donors) were the sole factor with a significant impact on migration rate. Automatic cell counting conveniently assessed the inhibitory effect of GRGDTP, an inhibitor of integrin-mediated cell adhesion. The method described here is rapid, does not require heavy equipment, and allows studies under serum-free conditions required to test molecules interfering with cell migration, in the course of the in vitro myogenic process.

Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the vasculature of oligodendrogliomas.

Vascular endothelial growth factor (VEGF) appears to be implicated in tumour angiogenesis. In the present study immunohistochemical expression of VEGF was evaluated in 34 oligodendrogliomas (13 grade II, 21 grade III [WHO]). VEGF immunoreactivity was found in 31 of 34 cases. Expression of VEGF was observed in endothelial cells and some vascular smooth muscle cells, but not in neoplastic oligodendrocytes. Vessel counts, percentages of VEGF-positive vessels and vessels with vascular endothelial proliferation were assessed. The degree of VEGF labelling and vascular-endothelial proliferation in each vessel were evaluated using a 3 degree intensity score. Expression of VEGF was higher in grade III than in grade II oligodendrogliomas as assessed by percentage of VEGF positive vessels (55.8 +/- 29.2% vs 17.0 +/- 19.0% [P < 0.001]) and by VEGF immunostaining intensity (1.90 +/- 0.60 vs 0.90 +/- 0.40 [P < 0.001]). VEGF expression did not correlate with vessel density. Intensity of VEGF expression correlated positively with that of vascular-endothelial proliferation in grade III tumours (r = +0.47 [P < 0.05]). The percentage of VEGF positive vessels showed some degree of positive correlation with the percentage of vessels showing vascular-endothelial proliferation (r = +408 [P < 0.10]). Within individual grade III tumours 67.5 +/- 29.6% of all vessels with vascular-endothelial proliferation were VEGF-positive and 31.0 +/- 20.5% of all VEGF-positive vessels showed no evidence of vascular-endothelial proliferation. We conclude that (i) expression of VEGF is observed in the vasculature of oligodendrogliomas; (ii) marked expression of VEGF is observed in grade III oligodendrogliomas; (iii) VEGF may be one of the interrelated causative stimuli acting in concert to induce vascular-endothelial proliferation.

Changes in the responsiveness of the colonic smooth muscle to carbachol in Hirschsprung's disease.

In vitro experiments were performed on smooth-muscle strips cut out in longitudinal and circular direction from the colon of Hirschsprung's patients who underwent an operation and from patients operated on for tumors of the sigmoid colon and rectum, serving as controls. The changes in the contractile activity of the smooth-muscle strips after carbachol applied cumulatively were examined and dose-response curves were plotted. The EC50 values for the circular strips from the aganglionic part of the colon were 6 x 10(-7) M in Hirschsprung's patients and 3.7 x 10(-8) M in control patients: the pD2 values were 6.23 and 7.43, respectively. This showed that the affinity of cholinoreceptors in the aganglionic part of the colon for carbachol was 16 times lower in Hirschsprung's patients as compared to control patients. The EC50 values for longitudinal strips from the ganglionic part of the colon were 2.6 x 10(-7) M in Hirschsprung's patients and 4 x 10(-8) M in control patients; pD2 values were 6.6 and 7.4, respectively. The affinity of cholinoreceptors in the smooth muscle of the ganglionic part of the colon for carbachol was also decreased (nearly 6.5 times) in Hirschsprung's patients as compared to controls.