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.

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.

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.

Immunological alterations induced by adjuvant treatment of postoperative colon carcinoma Duke's B or C with levamisole in combination with 5-FU.

BACKGROUND: Postoperative 5-FU combined with levamisole increases 5 year survival in colon cancer patients (Duke C) by 30% (1). In order to investigate the potential immunological mechanism, we determined lymphocyte subtypes and markers of immune activation in 22 patients before and during one year of postoperative adjuvant treatment. METHODS: Before and regularly during treatment, according to the scheme described by Moertel (1), major lymphocyte subsets were quantified by flow cytometry. Serum neopterin, soluble IL2-receptors, beta 2-microglobulin, TNF-alpha and interferon-gamma were determined by Elisa. RESULTS: The CD4/CD8 ratio increased significantly after levamisole was added to the treatment, as did the levels of soluble IL2-receptors. The percentages of T-cells expressing the interleukin 2 receptor followed a similar trend. The levels of neopterin tended to decrease during the combined treatment course. This was paralleled by a progressive fall in the proportion of T-cells expressing HLA-DR. CONCLUSIONS: The treatment induced significant and consistent alterations in major immunological mediators and lymphocyte subtypes. It remains to be established whether these changes are related to the therapeutic effect.

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.

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.

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.

Levamisole in the treatment of cancer: anything new? (Review).

Recently, levamisole combined with 5-FU was shown definitively to increase the survival of patients after surgery for colon cancer (9, 10). In the light of these findings the experimental and clinical findings with levamisole in the field of oncology are reviewed. The conclusion is reached that levamisole has proven activity, although its mechanism of action remains elusive. The progress that has been made in our understanding of immunology in general, the remarkable advance in experimental and diagnostic tools and the recent emergence of new indications of levamisole's interaction with immunosuppressive factors and interleukins all together warrant sufficient optimism to engage in a renewed clinical and experimental research effort. The outcome may be rewarding not only in terms of optimised treatment of patients with levamisole but also a more rational search for more potent successors may become feasible.

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.

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.

Nanovid microscopy.

By combining small colloidal gold probes with video-enhanced quantitative microscopy, the intracellular dynamics of specific proteins in living cells can now be studied.

Erbulozole (P.I.N.N.) (R 55 104) encapsulated into cyclodextrins: has it a combined antitumoral and radioprotective potential?

The clinically applicable formulation of the microtubule inhibitor erbulozole (R 55 104), dissolved into an aqueous hydroxypropyl-beta-cyclodextrin solution (designated as R 55 104-CYCLO), exerts a similar effect on growth delay of subcutaneous MO4 fibrosarcomas in mice, with or without 10 Gy gamma-irradiation given locally to the tumors 2 h later, compared to R 55 104 in water. The drug concentration can be reduced from 80 mg/kg to 5 mg/kg without affecting the activity of this particular drug-radiation combination. Furthermore, 80 mg/kg R 55 104-CYCLO show a radioprotective effect when given 2 h before total body irradiation of non-tumor bearing mice. A radiation dose of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Gy respectively was given resulting in a LD50(30) of 5.97 Gy for the irradiated mice and 7.65 Gy for the drug-radiation treated animals (Dose Effect Factor = 0.78). Therapeutic implications of both observations are discussed.

Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.

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.

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.

Detection of gold probes with video-enhanced contrast microscopy: nanovid microscopy.

This contribution describes a new microscopic technique aimed at visualizing colloidal gold particles of 20-40 nm diameter as dynamic markers at the light microscopic level. The technique, called nanovid microscopy, is based on the use of contrast enhancement by video techniques and digital image processing. Two applications on living cells are discussed. The first is the receptor-mediated endocytosis of the transferrin receptor, which for the first time can be followed in real time. A second application documents the motion of cell-surface glycoproteins on PTK2-cells. In addition, nanovid microscopy allows the number of gold particles in immunogold staining to be quantified easily. With this tool, the translocation of specific molecules in living cells becomes one of the most enjoyable activities to observe.

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling.

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.

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.