Maria Jonsson

Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil

Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [3H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, α-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer.

Presence of a marked nonneuronal cholinergic system in human colon: Study of normal colon and colon in ulcerative colitis

Background: The body has not only a neuronal but also a nonneuronal cholinergic system. Both systems are likely to be very important, particularly in inflammatory conditions. The patterns and importance of the nonneuronal cholinergic system in patients with ulcerative colitis (UC) are largely unknown. Methods: The colons of UC and non‐UC patients were examined for expression patterns of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and the muscarinic receptor of the M2 subtype. Results: ChAT and VAChT immunoreactions and mRNA reactions for ChAT were detected in epithelial and endocrine cells, in cells in the lamina propria, and in blood vessel walls. Furthermore, a marked M2 immunoreaction was noted for epithelium, blood vessel walls, and smooth musculature. ChAT and VAChT immunoreactions were significantly higher in endocrine and epithelial cells, respectively, in non‐UC mucosa than in UC mucosa. On the other hand, there was a tendency toward higher M2 levels in epithelium of UC patients. Conclusions: There is a pronounced nonneuronal cholinergic system in the colon, which has previously been ignored when discussing cholinergic influences in UC. Furthermore, it is evident that certain changes in the nonneuronal cholinergic system occur in response to inflammation/derangement in UC. Cholinergic effects in the colon can be considered to be related not only to nerve‐related effects but also to effects of acetylcholine from nonneuronal local cells. Thus, the recently discussed phenomenon of a “cholinergic antiinflammatory pathway” in the intestine may have a pronounced nonneuronal component.

Substance P and the neurokinin-1 receptor in relation to eosinophilia in ulcerative colitis.

Substance P (SP) has been implicated in the pathophysiology of ulcerative colitis (UC) and it has been suggested that blocking of its effect would be advantageous in this disease. Eosinophils have also been implicated in the pathophysiology of UC. In the present study, specimens from the sigmoid colon of UC patients were investigated by the use of antisera against SP and the neurokinin-1 receptor (NK-1R) and staining for demonstration of eosinophils. The degrees of SP innervation and NK-1R immunoreaction, as well as the levels of eosinophil infiltration, varied between different patients. Interestingly, NK-1R immunoreaction in the epithelium was often seen to be the most marked where there were numerous eosinophils in the underlying mucosa and where the mucosa showed a marked morphologic derangement. The observations suggest that there are marked fluctuations in effects of SP and eosinophils during the disease. The infiltrating eosinophils may be involved in the destruction of the mucosal tissue. Furthermore, for the majority of cases where there is marked derangement of the mucosa, it is apparent that there is an upregulation of the NK-1 receptor in the epithelium in parallel with the infiltration of the eosinophils.

Pachymetric evaluation prior to laser in situ keratomileusis

Purpose: To determine whether deviations in the localization of the corneas thinnest point or the magnitude and localization of posterior corneal ectasia is associated with deviations in the spherical equivalent, the astigmatism, or the magnitude of an anterior corneal ectasia and whether corneas at risk for iatrogenic keratectasia can be identified without a pachymetry map of the cornea. Setting: University hospital eye clinic. Methods: Three hundred eight eyes of 156 healthy volunteers with various refractive errors were examined with Orbscan II and autorefractometer–keratometer. The corneal thickness was registered at the fixation point, at the geometrical center, and at the thinnest point of the cornea. Keratometry and refraction were determined for all subjects. Results: The thinnest point of the cornea was predominantly located in the inferotemporal quadrant, and was significantly thinner than the fixation point (539.6 ± 35.8 μm and 548.0 ± 35.4 μm, respectively, P<.001). Interestingly, the larger this difference was, the longer the distance between these points. No relationship was found between the refractive or external surface measurements and the internal surface measurements. Conclusions: The absence of a clear relationship between the shape of the anterior corneal surface or the refractive error, and the shape of the posterior corneal surface, necessitates a thorough pachymetric evaluation of the cornea before a laser in situ keratomileusis procedure, with special attention to the inferotemporal area.

Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains.

The major outer surface proteins of Lyme disease spirochaetes are differentially expressed in different isolates. Borrelia afzelii strain F1 expresses none, or very low amounts, of the OspA and OspB proteins. To elucidate the mechanisms that control the expression of these abundant surface proteins the ospAB operon of B. afzelii F1 was cloned, sequenced and compared to the previously sequenced ospAB operon of B. afzelii ACAI and Borrelia burgdorferi B31. The two B. afzelii strains showed almost 100% identity at the DNA level, although Coomassie-stained gels and Western blot analyses showed significant variation in the Osp protein content. Transcriptional analysis revealed that the amount of ospAB mRNA produced in B. afzelii F1 varies more than the amount of protein, suggesting that the expression of OspA and OspB proteins is regulated at both the transcriptional and the translational level. Furthermore, the inverse relationship between the transcription of ospC and the ospAB operon could indicate coregulation of these separately encoded operons.

New aspects concerning ulcerative colitis and colonic carcinoma: Analysis of levels of neuropeptides, neurotrophins, and TNFalpha/TNFreceptor in plasma and mucosa in parallel with histological evaluation of the intestine

Background: The levels of neuropeptides, neurotrophins, and TNFalpha (TNF&agr;)/TNF receptor in plasma and mucosa for patients with ulcerative colitis (UC) and colonic carcinoma, and concerning plasma also for healthy controls, were examined. Moreover, the relationships between the different substances and the influence of mucosal derangement on the levels were analyzed. Methods: The levels of VIP, SP, CGRP, BDNF, NGF, and TNF&agr;/TNFreceptor1 were measured using ELISA/EIA. Results: Patients with UC demonstrated the highest levels of all analyzed substances in plasma, with the exception of BDNF. However, there were differences within the UC group, patients treated with corticosteroids, and/or nonsteroid antiinflammatory/immunosuppressive treatment having higher plasma levels than those not given these treatments. Patients with colonic carcinoma showed higher SP and TNFreceptor1 levels in plasma compared to healthy controls. Concerning mucosa, the levels of almost all analyzed substances were elevated for patients with UC compared to noncancerous mucosa of colonic carcinoma patients. There were correlations between many of the substances in both plasma and mucosa, especially concerning the 3 neuropeptides examined. There were also marked associations with mucosa derangement. Conclusions: Via analysis of correlations for the respective patients and via comparisons between the different patient groups, new and original information was obtained. Interestingly, the degree of mucosal affection was markedly correlated with tissue levels of the substances and the treatments were found to be of importance concerning plasma but not tissue levels of these. Combined plasma analysis of neuropeptides, neurotrophins, and TNFreceptor1 may help to distinguish UC and colonic carcinoma patients.

Subcutaneous implanted chambers in different mouse strains as an animal model to study genetic stability during infection with Lyme disease Borrelia

Tissue metal net cages were implanted subcutaneously in BALB/cJ and C3H/Tif mice as an experimental model of Borrelia burgdorferi infection. B. burgdorferi sensu stricto strain Sh2-82 could be isolated up to 14 weeks after the inoculation. However, a significant difference in infectivity between the two mice strains was observed. C3H/Tif mice were more susceptible to developing chronic B. burgdorferi s.s. infections than BALB/cJ mice. Although a B. burgdorferi infection was established, no rearrangements in the ospA and ospB genes were observed in any of the infected mice.

Decrease in binding for the neuropeptide VIP in response to marked inflammation of the mucosa in ulcerative colitis

Abstract:  The neuropeptide vasoactive intestinal peptide (VIP) is involved in the neuroimmunomodulation of the intestine. In the present study, specimens from the sigmoid colon of ulcerative colitis (UC) and non‐UC patients were examined for immunohistochemistry and in vitro receptor autoradiography. Marked occurrence of VIP binding was observed in the mucosa. However, there were very low levels of binding in areas showing pronounced inflammation/derangement. The study shows that marked derangement of the mucosa leads to a distinct decrease in VIP binding. Thus, it is possible that a decrease in trophic and anti‐inflammatory VIP effects occurs in areas exhibiting a very marked inflammation.