Mechanisms of osteoclastogenesis inhibition by a novel class of biphenyl-type cannabinoid CB(2) receptor inverse agonists.

The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.

Natalizumab: new drug. Multiple sclerosis: risky market approval.

(1) In relapsing-remitting multiple sclerosis, the standard therapy (other than symptomatic treatment) is interferon beta. It prevents about one exacerbation every 2.5 years but has no demonstrated effect on the progression of disability. However, interferon beta can cause serious adverse effects. (2) Natalizumab, an immunosuppressant, has been approved for first-line treatment of patients with "aggressive" multiple sclerosis (with frequent exacerbations) and for second-line treatment after failure of interferon beta. (3) In first-line treatment, natalizumab has not been compared with interferon beta. In a double-blind placebo-controlled trial involving 942 patients who were treated for 2 years, natalizumab prevented about 1 exacerbation every 2 years (0.24 versus 0.73 exacerbations per year). A retrospective subgroup analysis suggested that efficacy was better in patients with aggressive disease. As this was a post-hoc subgroup analysis, this exploratory hypothesis requires further testing. Natalizumab appeared to slow the progression of disability, but this result is undermined by the small percentage of patients who had an exacerbation (18% versus 27%). (4) In second-line treatment, a combination of natalizumab and interferon beta (rather than natalizumab monotherapy) was compared with interferon beta in 1171 patients in whom interferon beta had failed. The combination prevented about one exacerbation every 2.5 years. It is not known whether a combination of natalizumab and interferon is more effective than natalizumab alone. (5) Three cases of progressive multifocal leukoencephalopathy occurred during clinical trials, two of which were fatal. The risk of this viral infection, which is usually symptomatic only in severely immunosuppressed patients, was estimated at about 1 case per 1000 patients on natalizumab. (6) Little is known of the risks of long-term treatment with natalizumab, especially the risks of infections and cancer. (7) During two years of treatment, 6% of patients developed persistent anti-natalizumab antibodies, leading to reduced efficacy and a higher incidence of reactions during the infusion, as well as hypersensitivity reactions. (8) In practice, given the poorly assessed and potentially fatal risks of long-term treatment with natalizumab, the limited improvement in efficacy does not justify the use of natalizumab other than in comparative trials.

Leukocyte migration in acute colonic inflammatory bowel disease: comparison of histological assessment and Tc-99m-HMPAO labeled leukocyte scan.

UNLABELLED: Noninvasive leukocyte scintigraphy for assessment of localization, extent, and degree of active inflammation in acute colonic inflammatory bowel disease have been shown to correlate well with endoscopy. This study compared findings of mucosal leukocyte migration assessed histologically with those of technetium 99m hexamethylpropylene-amineoxime-labeled leukocyte scintigraphy. PATIENTS AND METHODS: Twenty-one patients hospitalized because of a first attack or a relapse of known inflammatory bowel disease were investigated using leukocyte scintigraphy followed by total colonoscopy with multiple biopsies within 24 h. Histological interpretation focused on the degree of segmental mucosal leukocyte infiltration. RESULTS: Fourteen patients with ulcerative colitis (UC) and seven with colonic Crohn's disease (CD) were included. With the use of histology as the reference method, maximal proximal disease extent was correctly assessed by the leukocyte scan in 11 patients (8 with UC, 3 with CD), although the rectal involvement was not visualized in 5. In seven patients, the extent assessments almost matched (+/- one segment), and in the remaining three patients (two UC, one CD) the scan grossly misinterpreted active histological inflammation (> or = +/- two segments). In patients with UC, the sensitivity, specificity, and diagnostic accuracy concerning the extent of inflammation were 0.84, 0.79, and 0.83, respectively. In patients with CD, the sensitivity was 0.79, and the diagnostic accuracy was 0.78. The relative leukocyte scan activity score was less concordant with the degree of mucosal leukocyte infiltration but still significantly correlated (r = 0.616, p < 0.0001 in UC patients and r = 0.441, p < 0.003 in CD patients). CONCLUSION: Images created by the technetium 99m hexamethylpropylene-amineoxime-labeled leukocyte scan in acute colonic inflammatory bowel disease correlate to mucosal leukocyte migration in terms of proximal disease extent and, to a lesser degree, also to the intensity of mucosal inflammatory infiltration.

Bovine leukocyte adhesion deficiency: in vitro assessment of neutrophil function and leukocyte integrin expression.

Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders.

[Immunologic assessment of preparations of the RS virus at the stages of its purification and concentration]. / Immunologicheskaia otsenka preparatov RS-virusa na étapakh ego ochistki i kontsentratsii.

The work demonstrates the immunogenic potency of some preparations of RS virus, differing in the degree of their purification and introduced by multiple intranasal administration. The immunogenic potency of these preparations was manifested by the synthesis of antibodies determined in the coagglutination test (the specificity of these antibodies was confirmed in the reaction of the neutralization of RS virus), as well as by sensitization determined in the leukocyte migration inhibition test. Immunization with concentrated virus was more effective than immunization with the preparation of virion fraction. The intranasal administration of live RS virus to mice induced the appearance of a morphologically determined disease which was not prevented by the development of immune response accompanying the disease. The conclusion was made that further improvement in the system of testing the preparations of RS virus was necessary.

Cross-reactivity of Mycobacterium leprae and M. tuberculosis and the implications for assessment of in vitro T cell function in leprosy patients.

The cross-reactivity in vitro between Mycobacterium leprae and M. tuberculosis was studied in 41 Aboriginal Australians with leprosy, 78 uninfected contacts of leprosy patients and 38 control individuals. A vigorous T cell response to epitopes cross-reactive between these two mycobacteria was found for healthy uninfected contacts or non-contacts (controls) of leprosy patients, but not for the patients themselves. The data suggest that a vaccine based on antigen shared between M. leprae and other mycobacteria is unlikely to be useful in preventing leprosy. Further studies of responses in vitro to purified T cell-reactive antigens would be useful in designing newer vaccines for more widespread field studies of leprosy prevention.

Assessment of neutrophil migration, phagocytosis and bactericidal capacity in neonatal foals.

Comparison of neutrophil function was made between 8 clinically normal pony foals (3 to 7 days of age), and their dams. Random migration, stimulated migration to zymosan-activated serum, bacterial phagocytosis and bactericidal capacity of neutrophils were determined in vitro. Random migration was greater (P less than 0.01) and stimulated migration was less (P less than 0.01) in foals than in their dams. Bacterial phagocytosis and bactericidal capacity of neutrophils were not different (P greater than 0.05) between foals and mares. Results of this study suggested that neonatal foals have altered neutrophil locomotion, when compared to their dams.

[Assessment of the effect of prospidin and cyclophosphane on the indices of the T and B immune responses in vitro]. / Otsenka vliianiia prospidina i tsiklofosfana na pokazateli T- i B-immunnogo otveta in vitro.

Prospidin was shown to produce a decrease of receptors on T- and B-lymphocytes and T-subpopulations, to inhibit migration of leucocytes under the influence of the antigenic stimulus, to reduce the cytopathic activity of lymphocytes and the level of secreted immunoglobulins of the main classes. The degree of prospidin immunodepressive effect is compared with that of cyclophosphane.

Macrophage spreading inhibition test as an alternate method for in vitro assessment of cell-mediated immunity against Entamoeba histolytica.

During cellular immune responses sensitized lymphocytes release a number of mediators collectively known as 'lymphokines'. The assaying of macrophage spreading inhibition (MSI) factor released by sensitized lymphocytes was employed as an alternative procedure for in vitro detection of cell-mediated immunity (CMI) in animals immunized with amoeba antigen preparation. The MSI test was compared with macrophage migration inhibition test. A well defined CMI was detectable in animals immunized by amoebic antigen in combination with Freund's complete adjuvant (FCA). Macrophage spreading was found greatly inhibited when peritoneal exudate cells from these animals were cultured in the presence of specific antigen. The optimal time for the development of typical reaction was 30 min. Incubation for a longer time resulted in macrophage clumping.

Assessment of cell-mediated immunity against coxsackievirus B3-induced myocarditis in a primate model (Papio papio).

Delayed hypersensitivity and myocarditis were induced in five baboons (Papio papio) after inoculation with myocarditic murine coxsackievirus B3 (CVB3m). By means of the [3H]thymidine incorporation assay and the agarose droplet cell migration inhibition assay, specific cell-mediated immunity was detected in all five animals against viral antigens and/or KCl-extractable soluble antigens extracted from the heart of a CVB3m-inoculated baboon. KCl-extracted antigens from normal baboon heart tissues, as well as KCl-extracted antigens from spleen or liver tissues from a CVB3m-inoculated baboon, failed to stimulate baboon peripheral blood lymphocytes or inhibit the migration of baboon peritoneal exudate cells. The appearance of dissimilar antigen(s) in extracts of heart tissues from CVB3m-inoculated baboons which did not contain infectious CVB3m parallels the appearance of novel KCl-extractable antigen(s) induced by CVB3m in murine heart tissues (Paque et al., J. Immunol. 120:1672-1678). The animal model described represents the first systematic evaluation of cell-mediated immunity in CVB3m-induced myocarditis in primates and suggests that a similar phenomenon may occur in humans.

Assessment of cellular immune response to cancer of the breast.

Cellular immune competence and cell-mediated immunity to tumor antigens have been studied in patients with breast cancer. Some patients have been shown to have depressed lymphoproliferative responses to phytohemagglutinin and in mixed leukocyte culture. In some cases, this depression appeared attributable to suppressor cells. Many patients with breast cancer had a cellular immunity to extracts of autologous or allogenetic tumors, as detected by lymphoproliferation and leukocyte migration inhibition assays. In addition, some breast cancer patients reacted to antigens associated with murine mammary tumor virus. Some of the tests for cellular immunity have revealed correlations with clinical course and, therefore, may be of use in the management of patients with breast cancer.

Assessment of host immune status during progressive growth and after excision of DNA virus (simian virus 40) tumors in hamsters: comparison of tumor-specific and tumor-unrelated parameters of immune responsiveness.

The kinetics of cell-mediated immunity to simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were compared to the kinetics of tumor-unrelated parameters of immune responsiveness in the assessment of the immune statuses of inbred MHA/SsLAK hamsters during the course of progressive syngeneic SV40 tumor growth and after tumor excision. With the use of the tumor cell neutralization test in vivo and the macrophage migration inhibition assay in vitro, specific cellular immunity to SV40 TSTA was detected by 4 days after tumor cell inoculation, when the tumor was small. This tumor-specific immune response was no longer detected at 7 days after tumor cell inoculation, when the tumor had reached a mean diameter of 12.5 mm, but it returned by 14 days after surgical excision of the tumor. The patterns of host responsiveness to mitogens in spleen cells derived from tumor-bearing animals or from tumor-excised animals generally showed little or no correlation with the kinetics of tumor-specific cellular immunity. The kinetics of the humoral immune response to murine erythrocytes, as determined by hemagglutination assays, correlated much more closely with the kinetics of tumor-specific immunity than did the responses to mitogens. IgG antibody (T-dependent) responses were more affected by progressive tumor growth than were IgM antibody (T-independent) responses. The data suggest that results of tests with the use of tumor-unrelated parameters of immune responsiveness for the assessment of the immune status of cancer patients should be interpreted with caution.