HIV-associated dysfunction of in vitro IL-12 production depends on the nature of the stimulus and on the CD4 T-cell count of the patient.
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Blood Cell Count↗
Biomedical subjects
M De Brabander
Publications and source records attributed to M De Brabander.
Measurement of in vivo rectal mucosal cytokine and eicosanoid production in ulcerative colitis using filter paper.
BACKGROUND: Excessive mucosal generation of cytokines and eicosanoids has been reported in vitro in ulcerative colitis (UC) using traumatising biopsy techniques, and in vivo using time consuming rectal dialysis. AIMS: To validate a simple filter paper technique to profile rectal mucosal production of cytokines and eicosanoids in vivo in patients with UC compared with controls. PATIENTS: Forty one patients with UC (21 with active disease) and 16 controls were studied. METHODS: In vitro, recovery of known concentrations of cytokine or mediator applied to filter papers was measured by ELISA following incubation in buffer. In vivo, patients and controls had filter papers apposed to the rectal mucosa briefly through a rigid sigmoidoscope. Filter papers were then incubated prior to assay by ELISA. RESULTS: In vitro validation studies showed that the filter paper technique could be used to measure mucosal release of interleukin-1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), thromboxane B(2) (TXB(2)), and prostaglandin E(2) (PGE(2)), but not interferon gamma (IFN-gamma). Mucosal release of IL-1beta, TNF-alpha, TXB(2) and PGE(2) were significantly increased in active UC (p=0.001) and correlated directly with disease activity (p=0.02). CONCLUSIONS: The filter paper technique confirmed increased rectal mucosal release of cytokines and eicosanoids in UC, in proportion to disease activity. The simplicity, safety and speed of the technique make it a practicable option for use in the outpatient clinic to study the pathogenesis of inflammatory bowel disease, and potentially its response to treatment.
Adult↗
Unchanged levels of interleukins, neopterin, interferon-gamma and tumor necrosis factor-alpha in cerebrospinal fluid of patients with dementia of the Alzheimer type.
Several histopathological studies suggest that amyloidogenesis in dementia of the Alzheimer type is accompanied by activated glia and glia-derived cytokines, leading to chronic, self-propagating, cytokine-mediated molecular and cellular reactions. As studies regarding inflammatory changes in cerebrospinal fluid of patients with dementia of the Alzheimer type has been inconclusive, we set up a prospective study to assess cerebrospinal fluid levels of interleukin-1beta, interleukin-6, interleukin-10, interleukin-12, soluble interleukin-2 receptor, interferon-gamma, tumor necrosis factor-alpha and neopterin in 20 patients with dementia of the Alzheimer type and 20 age- and sex-matched controls. Comparing both groups, no significant differences in concentrations and specific activities could be revealed. An additional 22 patients were included to enlarge the study population. No statistically significant differences were shown comparing patients (n=42) with the control group (n=20). We conclude that the immune-mediated inflammatory changes found in histopathological studies are not reflected in cerebrospinal fluid of patients with dementia of the Alzheimer type. Probably, cytokine production appears very localized in the central nervous system, not allowing representative detection in cerebrospinal fluid. Further studies assessing cytokine levels in various regions of central nervous system of patients with dementia of the Alzheimer type will be of interest to confirm this hypothesis.
Aged↗
Virological and immunological analysis of a triple combination pilot study with loviride, lamivudine and zidovudine in HIV-1-infected patients.
OBJECTIVE: To compare two antiretroviral regiments, loviride plus lamivudine (3TC) plus zidovudine (ZDV) (triple combination) and loviride plus ZDV (double combination) in terms of pharmacokinetic interactions, tolerability, safety, and immunological and virological efficacy. STUDY DESIGN: An open, case-controlled, pharmacokinetic and 24-week continuous treatment pilot study. PATIENTS: Twenty p24 antigen-positive patients, 10 per treatment group, were matched according to p24 antigenaemia less or more than 100 pg, CD4 count less or more than 150 x 10-(6)/l, and gender. Eight out of 10 cases and seven out of 10 controls had received previous antiretroviral therapy. RESULTS: No clinically relevant pharmacokinetic interactions were observed. Both treatment combinations were well tolerated. Median absolute and percentage CD4 count increases above baseline were more pronounced in the triple combination arm than in the double combination arm. Median p24-antigen and plasma viraemia level decreases below baseline were more pronounced in the triple combination arm. The M(184)I/V mutation was detected in all plasma samples of triple combination patients examined at week 12. Mutations conferring resistance to loviride and ZDV were found in a significant subset of patients in both treatment arms. CONCLUSIONS: Both combination regimens have an excellent safety/tolerability profile, but a higher level of in vivo efficacy is achieved by the triple combination, despite genotypic changes conferring resistance to one or all of these agents. The conclusions drawn are limited by small population size and the heterogenous pretreatment history. However, they support the validity of and strongly encourage a rationally designed multidrug combination approach to HIV therapy.
Acetamides↗
Evaluation of the efficacy and tolerance of R 018893, R 089439 (loviride) and placebo in asymptomatic HIV-1-infected patients. Loviride Collaborative Study Group.
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Acetamides↗
A simple and rapid flow cytometric method to measure lymphocyte activation in HIV+ subjects. Diminished response to pokeweed mitogen in early disease.
It was our objective to investigate the effect of asymptomatic infection with HIV on the expression of lymphocyte activation markers after stimulation with mitogens. Whole blood cultures were made of HIV+ and HIV- subjects (29 asymptomatic HIV-1-infected subjects and 33 apparently healthy normal volunteers). At various times after stimulation with concanavalin A (Con A), anti-CD3 and pokeweed mitogen (PWM), the expression of activation markers (CD25 and HLA-DR) and the blastogenesis were quantified by flow cytometry. The flow cytometric quantification of the expression of activation markers and blastogenesis in whole blood cultures provided an easy and safe alternative to thymidine incorporation to assess lymphocyte responses in HIV+ subjects. Activation showed a tendency to be lower in the HIV+ subjects with all three stimulants. This difference with HIV- subjects was statistically significant only for stimulation with PWM after 4 days. Further investigations should be undertaken to show whether this functional impairment is related to disease progression and whether it can be influenced by effective therapy.
Adult↗
Sabeluzole, a memory-enhancing molecule, increases fast axonal transport in neuronal cell cultures.
Morphological rearrangements, such as synapse number changes, have been observed in the adult mammalian brain after various experimental paradigms of learning and behavioral experience. The role of axonal transport in the physical translocation of material during this form of brain plasticity has not been fully appreciated. We show here by quantitative video microscopy that sabeluzole (R58735), a new memory-enhancing drug in humans, effectively increases fast axonal transport in rat neuronal cell cultures. Long-term incubation (24 hr) with sabeluzole in the concentration range between 0.1 and 1 microM increases both velocity and jump length of saltatory movements maximally by 20-30% in embryonic hippocampal neurons. Acute treatment only increases the velocity by 15-20%. Furthermore, the inhibition of axonal transport by 0.1 mM vanadate in N4 neuroblastoma cells is reversed by 1 microM sabeluzole. Observations on the kinesin-induced microtubule mobility in a reconstituted system show a 10% enhancement by sabeluzole at an optimal concentration of 2 microM, but no increase in kinesin ATPase activity. To our knowledge, this is the first pharmacological compound shown to increase fast axonal transport. The mechanism of fast axonal transport enhancement is discussed as a rationale for new therapeutic treatment in neuropathology.
Animals↗
Levamisole plus 5-fluorouracil inhibits the growth of human colorectal xenografts in nude mice.
Fragments of human colorectal adenocarcinomas were inserted under the renal capsule of nude mice. The growth of these tumour grafts was significantly inhibited by the combination of 5-fluorouracil (5-FU) and levamisole. An alternating regimen of levamisole 2.5 mg/kg and 5-FU 20 mg/kg decreased the size of tumour implants by 33-59% and/or increased the number of macroscopically disappeared fragments in the combined group compared with ineffective monotherapy with saline, levamisole or 5-FU. This model could be valuable for investigating the mechanism of action of levamisole and to evaluate the effects of this adjuvant therapy in other oncological settings.
Adenocarcinoma↗
Study of the parameters of binding of R 61837 to human rhinovirus 9 and immunobiochemical evidence of capsid-stabilizing activity of the compound.
The binding of the antiviral compound R 61837 to human rhinovirus 9 (HRV 9) was studied quantitatively and compared with binding of R 61837 to HRV 9H, a semiresistant variant. For both strains, radiolabelled R 61387 bound to native particles only. The Kd values obtained by Scatchard analysis of saturation binding data were 37 nM for HRV 9 and 172 nM for HRV 9H, whereas the concentrations resulting in a 50% reduction of cytopathic effect were 42 nM and 840 nM, respectively. Reversibility experiments showed that 65% of the compound could be extracted with chloroform from HRV 9H but less than 5% could be extracted from HRV 9. Dissociation studies demonstrated that in the presence of excess unlabelled compound, the half-lives of the virus compound complex HRV 9 and HRV 9H were 385 and 15 min, respectively. The effect of this antirhinoviral compound on the formation of subviral particles induced by low pH or heat was also investigated. Rate zonal centrifugation experiments using [35S]methionine-labelled HRV 9 showed that binding of R 61837 protected the virus against heat (56 degrees C) and acid (pH 5.0) and that at the same concentration of R 61837 the semiresistant strain was stabilized to a lesser extent. This observation was confirmed immunochemically with nonneutralizing and neutralizing monoclonal antibodies. Both 80S and 130S subviral particles have C antigenic determinants, whereas native particles (150S) have been designated D. R 61837 prevented the switch from D to C antigenicity which can be induced by exposure of rhinoviruses to mild denaturing conditions. These findings indicate that the compound is able to prevent a conformational change of the capsid which may be a prerequisite for infection.
Antibodies, Monoclonal↗
Laminin binding and internalization by human and murine mammary gland cell lines in vitro.
We have studied the binding and internalization of Engelbreth-Holm-Swarm mouse sarcoma laminin labeled with colloidal gold (LN-G40) by human and murine mammary gland cell lines. Interactions between the LN-G40 probe and the cells spread on a glass coverslip were monitored with video-enhanced contrast microscopy (Nanovid). Transmission electron microscopy allowed the quantitation of the LN-G40 probe at various cellular locations. During the first 15 min, a homogeneous binding of LN-G40 probe to the cell surface was observed with all cell lines. This binding did not occur with gold particles that were not conjugated to laminin. Then, the LN-G40 probe began to cluster on the cell surface and was, during the following 20 h, internalized by pits that were not coated. In the cells, the LN-G40 probe sometimes showed saltatory movements along linear tracks. The LN-G40 probe was intracellularly found in vesicles, multivesicular bodies, cisternal structures, and lysosomes, suggesting the degradation of the internalized laminin. However, not all cell surface-bound LN-G40 probe was internalized after 20 h. Differences between the cell lines were quantitative, but no clear correlation could be made between migration of cells on laminin and internalization of laminin.
Animals↗
Serum and serotonin induce retraction of calf aortic smooth muscle (CASM) cells in vitro: inhibition by ketanserin, a 5-HT2 receptor antagonist.
Calf aortic smooth muscle (CASM) cells cultured in vitro at high cell density (4 x 10(4) cells/cm2) on bacteriological petri dishes in the presence of serum pile up in clusters and create open spaces in the monolayer. This phenomenon is clearly visible 6 days after plating and is markedly enhanced by the addition of fetal calf serum. Serotonin is essential for the serum-induced retraction since (1) dialyzed serum has no effect, (2) of all the vasoactive agents we tested, only serotonin induced a similar degree of retraction, and (3) the serum-induced retraction was completely blocked by preincubating the cells with serotonin 5-HT2 receptor blockers such as ketanserin and ritanserin but not by preincubation with adrenergic-alpha 1 blockers or histamine antagonists. Serotonin caused CASM cell retraction in a dose-dependent way, with a maximum effect at 10(-6) M. The serotonin-induced retraction was reversible in time and was effectively blocked by ketanserin (IC50 = 1.2 x 10(-9) M). It is therefore concluded that serotonin induces retraction of CASM cells, mediated by the serotonin 5-HT2 receptor.
Animals↗
Influence of the synthetic microtubule inhibitor erbulozole (P.I.N.N.) (R 55 104), a new tubulozole congener, and gamma irradiation on murine tumors in vivo.
Erbulozole (P.I.N.N.) (R 55 104) is a more water soluble congener of the synthetic microtubule inhibitor tubulozole (R 46 846) exhibiting a reversible antimicrotubular activity in vitro at a dose (1.56 x 10(-8) M) which is at least 10-fold lower. The compound also has an antiinvasive potential and shows antitumoral effects both in vitro and in vivo when administered appropriately. Eighty mg/kg R 55 104, given orally 6 h before or 3 h after radiotherapy, displays a prominent interactive effect with 10 Gy gamma irradiation in subcutaneous murine tumors which is similar to 160 mg/kg tubulozole administered 6 h before 10 Gy. The enhancing effect is also observed in a clinically relevant radiation dose fractionation schedule whereby eight fractions of 2 Gy each were pretreated 2 h before with 40 mg/kg R 55 104. Further study of this radiochemotherapeutic combination may lead to new clinical applications.
Animals↗
Interaction between the microtubule inhibitor tubulozole and gamma-irradiation in murine tumors in vivo.
The combined effect of the microtubule inhibitor tubulozole and gamma-irradiation has been investigated in vivo in subcutaneous MO4 fibrosarcomas and Lewis Lung carcinomas. A marked interactive effect on tumor growth was observed when 160 mg/kg tubulozole was orally administered before the tumors were treated with 10 Gy radiation. Dose dependency and optimal effect were obtained on tumor growth of MO4 tumor bearing animals when the drug treatment was given 6 hr prior to the irradiation. The optimal pretreatment time coincided with the time at which a peak mitotic index in the tumor tissue was observed. An enhancing effect is also noticed at other doses of radiation in MO4 tumors pretreated 6 hr before with 160 mg/kg tubulozole. The interactive effect is maintained in a clinically relevant dose fractionation schedule whereby 8 fractions of 2 Gy each were pretreated 6 hr before with 80 mg/kg tubulozole. Tubulozole-T, the stereo-isomer of tubulozole, neither exhibits any antimicrotubular action nor exerts an antitumoral effect on its own or in combination with gamma-irradiation. The possible mechanisms of interaction between tubulozole and gamma-irradiation in tumor tissue are discussed.
Animals↗
Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy.
Transferrin receptors labeled with the B3/25 monoclonal antibody-gold complexes were followed in living A431 cells by using video-enhanced contrast microscopy. Initially, the antibody-gold complexes bind to receptors which are freely mobile on the upper cell surface; they then become trapped at the inner margins of the peripheral lamellae and internalize. During endocytosis discrete gold-loaded vesicular elements first appear, and then, as they fuse, a heterogenous peripheral endosomal compartment forms. The endosomes from this compartment then begin to migrate centripetally through the cytoplasm in a saltatory way so that within 15 min gold label accumulates in a juxtanuclear endosome compartment. This compartment, which consists mainly of multivesicular bodies, is thus formed by the influx and retention of peripheral endosomal elements and their continued fusion in the juxtanuclear area. Although their overall migration is inward, saltating endosomes frequently reverse their direction of movement. As label builds up in the juxtanuclear area, small vesicles containing gold label continuously pinch off from the larger elements and migrate toward the cell periphery. Experiments with nocodazole and sodium azide show that the saltatory movements, the accumulation and retention of endosomes in the juxtanuclear area, and the separation of vesicles from endosomes are driven by a microtubule-associated, ATP-dependent, motility-generating mechanism. Analysis of the movements shows that although each individual vesicle saltation can occur unpredictably toward the centre or the periphery of the cell, a net centripetal flux is observed. Moreover, it is evident that the probability of migration toward and maintenance in the juxtanuclear area is related to the diameter of the vesicles. We propose a mechanism by which bidirectional saltation along microtubules forming a radial network may be instrumental in the selective concentration of large endosomes in the juxtanuclear area while small vesicles are left free to return to the periphery. This process may be responsible for the sorting of receptors and ligands destined either for intracellular degradation in juxtanuclear lysosomes or, alternatively, for recycling to the plasma membrane.
Alkaloids↗
In vitro activity of R 61837, a new antirhinovirus compound.
R 61837 or 3-methoxy-6-[4-(3-methylphenyl)-1-piperazinyl]pyridazine is a new and potent inhibitor of rhinoviruses at concentrations not inhibitory to HeLa cell growth. Different rhinovirus serotypes varied widely in their susceptibility to the antiviral agent. The MICs for 50% CPE reduction ranged from 0.004 to 15 micrograms/ml. The yields of the most susceptible serotypes were reduced by a factor of 1,000 to 10,000 after single round high multiplicity infections in presence of low concentrations of the compound. The inactivation of some but not all serotypes in a time-, concentration- and temperature-dependent way by R 61837 indicated a direct interaction between the drug and the viral particles. The antiviral activity of the compound was confirmed in the human target cells for rhinoviruses by experiments using nasal polyp explant cultures.
Animals↗
Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy.
We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.
Animals↗
The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell.
We describe a new approach to probe the molecular biology of the living cell that uses small colloidal gold particles coupled to specific ligands. They are visualized in cells by bright-field, video enhanced contrast microscopy. We describe the basic aspects of the technique and provide examples of applications to intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing. We also provide examples of the use of this approach in immunospecific labelling of cells and tissue sections.
Animals↗
Preembedding for localization by electron microscopy of cytoskeletal antigens in cultured cell monolayers using gold-labeled antibodies.
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Antibodies↗