Generation of cytokines in human visceral leishmaniasis: dissociation of endogenous TNF-alpha and IL-1 beta production.

The role of TNF-alpha in visceral leishmaniasis is ambivalent, the eventual outcome of this infection, cure or generalization, being determined by the relative amounts of cytokines produced in vivo. Since release, by monocytes/macrophages, of TNF-alpha and interleukins 1 beta (IL-1 beta) and IL-6 is important in both the induction and effector phases of the immune responses, these mediators were determined in sera and cell culture supernatants of seventeen L. donovani infected patients in Brazil. The results are compared to those of a local control group. Circulating immunoreactive TNF-alpha in patients (median, 140 pg ml-1) was increased ten-fold over controls (median 16 pg ml-1, p less than or equal to 0.0001). In contrast, serum IL-1 beta was less than 20 pg ml-1 in all patients, although detectable in sera of 3/16 Brazilian controls (chi 2 = 3.5, p less than 0.1). Mitogen induced in vitro release of IL-1 beta and IL-6 by patients' circulating mononuclear cells was significantly reduced, and the capacity of patients' peripheral monocytes for H2O2 generation in response to opsonized zymosan was significantly diminished. In the patients, serum TNF-alpha levels were inversely related to IL-1 beta release in vitro (rho = -0.57, p less than or equal to 0.01).

T cell phenotype alterations in hepatosplenic schistosomiasis mansoni normalize after chemotherapy.

Peripheral blood T cell phenotypes, CD3-induced mitogenesis and soluble IL 2 receptor and CD8 in sera were studied in intestinal and hepatosplenic Schistosomiasis mansoni before and three to six months after therapy with praziquantel. Fifteen pairs matched for intensity of infection were analyzed and compared with local, non-infected age-matched controls. CD3+ cell counts were lower in untreated hepatosplenic schistosomiasis (median 1040 cells/microliters; 95% confidence interval 608-1269) compared to controls (1534; 1264-1620). This difference was largely accounted for by immature CD1+/CD3-cells circulating in these patients (median 388/microliters, 252-474). The frequency of CD1+ T cells in circulation decreased drastically after chemotherapy. Similar, but less marked, alterations were seen in intestinal schistosomiasis. Lymphocyte proliferation initiated by agonistic anti-CD3 monoclonal antibody was severely impaired in hepatosplenic patients, who had suffered haemorrhagic complications, but not in the cases of incipient hepatomegaly. Soluble CD8 antigen circulated in increased amounts in hepatosplenic schistosomiasis. Remarkably, a negative correlation between CD3-induced mitogenesis and circulating levels of CD8 was noted in these patients. Whereas CD3-induced mitogenesis in hepatosplenic schistosomiasis normalized after therapy, circulating IL 2R and CD8 antigen in hepatosplenic patients still exceeded control levels. The results demonstrate disturbances of CD3 and CD8 expression and/or T cell maturation in hepatosplenic schistosomiasis. Imbalanced CD4/CD8 ratios and an increased IL 2R/CD8 turnover may reflect an inhibitory circuit within the T cell compartment.

Immunomodulation by thalidomide: systematic review of the literature and of unpublished observations.

Three decades of immunological investigations using thalidomide are reviewed. Both in vitro and in vivo investigations are in accordance with the clinical finding that thalidomide does not impede T-cell competence in the control of infection by mycobacteriae. The term immunosuppressant does not apply. The immunomodulatory effects of thalidomide are evident in a myriad of phenomenological changes, and a molecularly defined common denominator of these activities is not known at present. Critical assessment with the objective to account for the clinical activity of thalidomide in specific human diseases leads to a focus on effects of thalidomide on phagocytic leukocytes and endothelia. The former are responsive to thalidomide by modulation of cytokine synthesis in vitro and in vivo; this activity can be shown using monocyte-specific stimuli in peripheral blood mononuclear cells but also in other phagocytic cells like microglia. For technical reasons, endothelial cells have until now been tested primarily in vitro. However, there is solid evidence now from intravital microscopy that the induction of adhesivity in postcapillary venules by LPS is modulated by thalidomide. Altered surface antigen expression has been described on leukocytes obtained from humans and experimental animals treated with thalidomide, but convincing evidence is lacking for in vitro modulation of surface antigen expression on leukocytes (as opposed to the modulation of adhesion antigens on endothelial cells stimulated by LPS or exogenous TNF alpha in the presence of thalidomide). Therefore, in vivo redistribution is likely to account for some, if not all, changes in circulating leukocyte phenotypes. The immunopathological conditions most clearly responsive to thalidomide are vasculitic alterations of post-capillary venules either in the context of mycobacterial infection (in the case of erythema nodosum leprosum) or mucocutaneous aphths. In both instances (as in the majority of focal inflammatory lesions), leukocyte infiltration and cytokine responses, in particular TNF alpha, are present. Thalidomide acts clinically not only by palliation of existing lesions but also by prevention of recurrence. The mechanism operates in skin, mucosa and parts of the nervous system and is most readily explained by synergism of TNF alpha modulation and a separate point of action on leukocyte migration patterns.

Effects of thalidomide on the local Shwartzman reaction in mice and rabbits.

The Shwartzman reaction is an animal model displaying histopathological vasculitis phenomena. Extravasation and swelling due to increased vascular permeability and cellular infiltration, which are hallmarks of the Shwartzman reaction, were evaluated as leakage of i.v.-injected Evans Blue dye and by histological and immunohistological characteristics in rabbits and mice. (+/-)-Thalidomide, (-)-thalidomide, (+)-thalidomide and dexamethasone inhibited the increase of vascular permeability in the local Shwartzman reaction. Histologically, the intensity of the Shwartzman reaction was reduced. In mice thrombus formation and leukocytoclastic vasculitis was inhibited by (+/-)-thalidomide and (+)-thalidomide. ICAM-1 expression was markedly reduced after (+)-thalidomide injection. Thalidomide and dexamethasone pretreatment reduced Mac-1 expression on perivascular infiltrated granulocytes. The inhibitory effect of thalidomide on vasculitis of the Shwartzman reaction may thus be related to reduction of adhesion molecule expression.

Praziquantel in the treatment of hepatosplenic schistosomiasis: biochemical disease markers indicate deceleration of fibrogenesis and diminution of portal flow obstruction.

Serological indicators for disease activity and portal vein obstruction and/or deviation were assessed in 23 patients with hepatosplenic schistosomiasis before, and up to 18 months after, praziquantel treatment, as well as in 25 matched local controls. Cessation of egg-induced immunopathology was reflected by the return to normal of serum procollagen-III-propeptide and neopterin concentrations. Reversibility of portal vein pathology was indicated by normal clearance of cholylglycine in cases without signs of decompensating portal hypertension. In most patients with a history of ascites and/or haemorrhage, serum cholylglycine concentration remained pathological. The results provide evidence that the fibrogenic process ceases after specific chemotherapy, and that portal vein pathology regresses in a substantial proportion of hepatosplenic schistosomiasis cases.

Liver involvement in human schistosomiasis mansoni. Regression of immunological and biochemical disease markers after specific treatment.

Peripheral blood cholyglycine and procollagen-III-peptide were measured in 22 Zairean patients with hepatomegaly caused by S. mansoni before and after treatment with praziquantel. Circulating T-cell subsets and cutaneous in vivo delayed type hypersensitivity were assessed; serum neopterin and beta 2-microglobulin served as indicators for macrophage/lymphocyte activation. The results were compared to age and sex matched patients with S. mansoni infection limited to the intestinal tract and schistosomiasis free controls with equal socioeconomic background. Abnormal serum cholyglycine and neopterin levels and alterations of circulating T-cell subset frequencies were associated with hepatomegaly in schistosomiasis. Normalization of these parameters reflected a regression of egg-induced immunopathology as early as two months after specific chemotherapy. Serum procollagen-III-peptide concentrations rose significantly after treatment, suggesting release of propeptide previously incorporated without cleavage into tissue collagen. The combination of these biochemical and immunological parameters may allow assessment of the pathophysiological mechanisms responsible for liver disease in individual patients.

Presence of circulating anodic antigen in serum of Schistosoma intercalatum-infected patients from Gabon.

The presence of the schistosome circulating anodic antigen (CAA) in serum of Schistosoma intercalatum-infected patients from Gabon has been investigated using an enzyme-linked immunosorbent assay (ELISA). Blood samples were collected from 10 endemic controls, 29 patients which excreted viable S. intercalatum eggs in rectal mucosa and stool, six persons in which only non-viable eggs were found in rectal biopsy specimens and one person in which besides non-viable eggs a small number of viable eggs was found in the rectal biopsy specimen. CAA, a genus-specific antigen, could be demonstrated in 58.6% of the patients with S. intercalatum eggs in their stools. In comparison to S. mansoni infections, very light infections (0.6 eggs per gram faeces) could be detected by the ELISA. A strong correlation between parasite burden (eggs per gram faeces) and antigen-level (CAA-titer) was found (Spearman's rho = 0.65). Only one positive ELISA-results was found in the group with solely non-viable eggs in rectal tissue. As no false positive results were detected for the negative controls, the present results suggest, in accordance with results earlier obtained for schistosomiasis mansoni, that only in active S. intercalatum infections is antigen demonstrable.

Thalidomide suppresses T- and B-cell responses to myelin antigen in experimental allergic neuritis.

The effects of thalidomide and, for reference, dexamethasone on T- and B-cell functions were assayed in vitro in Lewis rats with experimental allergic neuritis induced by active immunization with bovine peripheral nerve myelin (BPM) and complete Freund's adjuvant. Thalidomide and dexamethasone at the concentration ranges 10(-5)-10(-7) g/ml and 4 x 10(-5)-4 x 10(-9) g/ml, respectively, both inhibited phytohemagglutinin (PHA)- and BPM-induced T-cell proliferation as well as levels of PHA- and BPM-reactive interferon (IFN)-gamma-secreting cells, reflecting the suppression of Th1-like cells. The effect of dexamethasone was most pronounced on PHA-induced T-cell proliferation and IFN-gamma secretion, whereas the effect of thalidomide was most pronounced on BPM-induced T-cell proliferation and IFN-gamma secretion. Thalidomide reduced the B-cell responses to both BPM and Mycobacterium tuberculosis purified protein derivative, but to a lesser extent than dexamethasone. The in vitro design described could be useful to evaluate compounds with putative immunomodulatory activities. The inhibitory effects of thalidomide on autoantigen-induced Th1-cell functions may warrant the use of this substance in T-cell-mediated autoimmune diseases.

Local skin reactivity after induction of Shwartzman reaction in rabbits.

A local Shwartzman response was elicited in rabbits by an intradermal injection of the Salmonella typhosa endotoxin lipopolysaccharide (LPS) followed 24 hours later by an intravenous challenge injection with zymosan. After the intravenous challenge, necrotizing vasculitis developed in the prepared skin sites which was characterized by microthrombi, accumulation of neutrophil granulocytes, fibrin deposition and extravasation of red blood cells. Evans' blue extravasation into the altered tissue was significantly reduced, and histologically, the intensity of the Shwartzman reaction in the skin was reduced by pretreatment with thalidomide and dexamethasone. The mechanism of reduction of an LPS-induced local Shwartzman reaction by thalidomide is discussed.

Thalidomide selectively modulates the density of cell surface molecules involved in the adhesion cascade.

The mode of action of thalidomide (THD) in clinical cases of vasculitis is still not clear. Expression of adhesion molecules on endothelial cell lines was therefore assessed in vitro. THD is capable of changing the density of tumor necrosis factor alpha (TNFalpha) induced ICAM-1 (CD54), VCAM-1 (CD106) and E-selectin antigens on HUVEC. Furthermore, modulation of L-selectin (CD62L) by THD can be demonstrated on human leukocytes in vitro. The molecules investigated are involved in the neutrophil-endothelial cell interaction and participate in the adhesion cascade. Blunting of cytokine induced up-regulation of these adhesion molecules may account at least in part for anti-vasculitic effects of thalidomide.

Extravasation of leukocytes assessed by intravital microscopy: effect of thalidomide.

OBJECTIVE AND DESIGN: Thalidomide is very effective in the treatment of idiopathic aphthous stomatitis, characterized by recurrent focal intramucosal leukocytic vasculitis. The mode of action of thalidomide in this clinical entity may include inhibition of the extravasation of leukocytes. Therefore, we studied the effect of thalidomide on different steps of leukocyte migration by intravital microscopy. MATERIAL: Male Syrian golden hamsters were used. TREATMENT: Leukocyte migration in buccal mucosa of the hamster cheek pouch was elicited by the local application of lipopolysaccharide (LPS, 20 micrograms/ml) or murine tumor necrosis factor-alpha (muTNF-alpha, 10 ng/ml). (+)-Thalidomide (20-200 mg/kg i.p.) was administered 60 min before the local application of LPS or muTNF-alpha. Dexamethasone (2 x 1.0-10 mg/kg i.p.) was administered 18 h and 60 min before topical LPS application. METHODS: The numbers of rolling, firmly adherent and migrating leukocytes were estimated by intravital microscopy up to 165 min after the topical applications of LPS or muTNF-alpha and evaluated by an interactive image analysis software. RESULTS: Thalidomide (20-200 mg/kg i.p.) dose-dependently inhibited LPS-stimulated perivenular leukocyte migration by up to 87 +/- 5% and muTNF-alpha-induced leukocyte migration by up to 78 +/- 4%. Dexamethasone (2 x 1.0-10 mg/kg i.p.) inhibited LPS-stimulated leukocyte migration by up to 85 +/- 13%. (+)-Thalidomide (200 mg/kg i.p.) inhibited LPS-stimulated rolling by 80 +/- 5% and reduced the number of firmly adherent leukocytes by about 40%. Dexamethasone (2 x 10 mg/kg i.p.) did not reduce the number of rolling leukocytes but inhibited leukocyte adherence by 72 +/- 9%. CONCLUSIONS: These results show that (+)-thalidomide predominantly inhibits leukocyte rolling and thus differs from the glucocorticoid dexamethasone. The inhibition of LPS- or muTNF-alpha-induced leukocyte extravasation by thalidomide may account for some of its clinical activities.

Antibodies of the IgE and IgG isotype, serum IgE and circulating immune complexes in schistosomiasis intercalatum.

Individuals in an advanced phase of infection by Schistosoma intercalatum in whom viable ova are still present in the rectal mucosa but not excreted in detectable quantity, can be distinguished clinically from a younger group of individuals excreting S. intercalatum ova in their faeces. This clear-cut distinction is underlined by findings in parameters of humoral response. The ratio of anticercarial to anti-adult worm antibodies was higher in the group excreting eggs with probably more recent infection. Levels of total serum immunoglobulin were higher in this group, with the exception of serum IgA, which was lower as long as eggs were being excreted. In a later stage of the disease, relatively more IgG seems to be bound in circulating immune complexes. It is postulated that these stage specific patterns of humoral immune response represent the evolution of the host-parasite relationship during the infection.

New approaches to the measurement of morbidity in intestinal schistosomiasis.

New approaches for the assessment of morbidity due to schistosomiasis infection include the substitution of egg counts by quantitative assays for circulating schistosomal antigens, simple parameters for global immune responsiveness and functional hepatic alterations, and ultrasonography as an imaging technique. Quantification of circulating schistosome antigens in sera of S. mansoni, S. haematobium or S. intercalatum infected patients by sensitive immunoassays provides an exact measure for the individual worm burden. The effect of chemotherapy on immunomodulation was assessed by a panel of cutaneous recall antigens. Non-specific cell-mediated immune responses returned to subnormal pretreatment levels in the event of reinfection. Immunological morbidity can be monitored in this way. Pathophysiological alterations of the liver in S. mansoni infection can be assessed by the determination of unconjugated bile acids in the systematic circulation. The regression of intrahepatic vascular lesions in schistosomiasis with hepato(spleno)megaly can be detected earlier by this parameter than by ultrasonography. Ultrasonography provides characteristic, if not pathognomonic, images of Symmers fibrosis, and may be of value in the long-term evaluation of hepatic fibrosis.

Effects of a new immunotherapeutic agent (CG5601) on endotoxin-induced uveitis.

CG5601 is a novel immunomodulatory substance showing anti-inflammatory properties comparable to thalidomide. To investigate the anti-inflammatory effects of CG5601 in endotoxin-induced uveitis (EIU) and to evaluate its influence on leukocyte-endothelium interaction, the anterior chamber inflammatory reaction was assessed and intravital fluorescence microscopy was carried out at 2, 4, 8 and 24 h. Lewis rats received an intraperitoneal injection of CG5601 (200 mg/kg b.w.) at the time of lipopolysaccharide injection. At 8 and 24 h, CG5601 inhibited the cell migration and protein concentration in the aqueous humor compared to untreated EIU (p < 0.0001). There was no significant difference between nontreated animals and vehicle controls. The treatment of CG5601 reduced the number of rolling leukocytes. At early time points (2 and 4 h), inhibition of rolling leukocyte flux was significant (p < 0.005). The rise of serum TNF-alpha levels in EIU at 2 h was reduced. CG5601 exerts potent anti-inflammatory effects in EIU.

Praziquantel compared to niridazole in schistosomiasis intercalatum therapy.

Praziquantel was compared to niridazole in schistosomiasis intercalatum in the Gabon. In a pilot study, 45 patients were randomly allocated to the two treatment groups. After treatment, 36 of these patients could be evaluated. Though the seven-day niridazole treatment showed significant reduction of egg excretion in all patients, cure rate was rather low. 9 out of 17 patients (53%) still excreted viable eggs of Schistosoma intercalatum. In contrast, patients treated with praziquantel in a one-day treatment (2 x 30 mg per kg) showed a persistent excretion of viable eggs in 3 out of 19 cases (16%). This difference was significant (p less than 0.025). Although the number of patients observed is rather small, results indicate that praziquantel is superior to niridazole in the treatment of schistosomiasis intercalatum both in view of application and side effects as well as curative potential.

Diagnostic value of rectal biopsy and concentration methods in Schistosomiasis intercalatum: quantitative comparison of three techniques.

The diagnostic value of the rectal biopsy was compared to a quantitative stool filtration and the MIF concentration technique in schistosomiasis intercalatum in the Gabon. The rectal biopsy was significantly more sensitive in proving the presence of ova of Schistosoma intercalatum than the stool filtration (p less than 0.001). However, if the two techniques were compared on the basis of the capability to detect viable eggs in rectal mucosa or stool, no difference in sensitivity could be found. The MIF concentration technique was significantly less sensitive than the stool filtration (p less than 0.01) and seems only of minor diagnostic value in schistosomiasis intercalatum. It could be shown that the relative sensitivities of the three techniques in detecting S. intercalatum eggs depended on the age of the examined population, as presence of viable eggs in rectal mucosa and excretion of eggs in stool was inversely correlated to the age of the patients.

5'-Substituted thalidomide analogs as modulators of TNF-alpha.

The synthesis of 5'-substituted thalidomide analogs is described. The amino acids 2 necessary to synthesize the target compounds were prepared by Michael reaction. Condensation of 2 with phthalic anhydrides followed by reaction with urea yielded 4 as diastereomeric mixtures. Furthermore glutethimide (5) was brominated by an improved method and the resulting compound 6 was reacted in several steps with sodium azide, hydrogen, and phthalic anhydride to give 8. In a similar manner, 6 was reacted with sodium azide and various phthalic anhydrides to give 9, 10, and 11. All final compounds were tested in vitro for their inhibitory activity on the release of TNF-alpha, using stimulated peripheral mononuclear blood cells (PBMCs). Compounds with an additional aromatic substituent in position 5' of the thalidomide molecule were more active than thalidomide. Compound 11 was able to reduce increased levels of IL-2 in vitro.