Adverse effects during specific oral tolerance induction: in home phase.

BACKGROUND: Specific oral tolerance induction (SOTI) is a promising approach for severe food allergies. There are little data in the literature regarding the home-phase of SOTI, not only with regard to type and frequency of adverse reactions but also regarding the most suitable treatment and protocol. AIMS: To define the incidence and severity of adverse reactions, possible risk factors, and the safety and effectiveness of the home-phase of an original SOTI protocol in a large group of children with severe cow's milk (CM) allergy, after the hospital "rush" phase. METHODS: The study was conducted by recording in-home phase adverse events, success and failure as reported by parents, and calling families. Adverse reactions were treated following the International Guidelines, arbitrarily modified by introducing nebulised epinephrine for respiratory reactions, oral beclomethasone for acute gastric pain and oral cromolyn for recurrent gastric pain. RESULTS: Out of 140 patients, 132 were contacted; eight were inaccessible (follow-up 2-84 months). The number of adverse reactions was 1 in every 100 doses. The reactions were treated with nebulised epinephrine (221 reactions), IM epinephrine (6 reactions), and other drugs. Patients with high specific IgE levels (greater than 100 kU(A)/L) and lower CM dose (less than 5 ml) at the end of in-hospital phase showed a higher risk both for number of reactions and use of nebulised epinephrine. CONCLUSIONS: The home phase of SOTI was characterised by a significant number of adverse reactions, mostly managed with an acceptable rate of side effects. Nebulised epinephrine played a pivotal role in respiratory reactions.

Diagnosed child, treated child: food challenge as the first step toward tolerance induction in cow's milk protein allergy.

BACKGROUND: Food challenge is required to assess tolerance in cow milk (CM) allergy. A positive challenge contraindicates the reintroduction of CM. Specific oral tolerance induction (SOTI) is a promising treatment. METHODS: All children admitted for a challenge were prospectively enrolled. To those tolerating between 2 and 150 ml a SOTI protocol was offered. Outcome, adverse reactions, parents' satisfaction were recorded. RESULTS: Out of 245 challenged patients, 175 reacted 122 out of 125, able to tolerate a minimum dose of 2 ml, underwent SOTI. After one year 75.4% were in an unrestricted diet, 16.1% tolerated between 5 and 150 ml, 8.5% stopped SOTI. Side effects were mild, parents' satisfaction was very high. CONCLUSIONS: The majority of children tolerating limited amounts of CM at the challenge acquires tolerance with SOTI without relevant side effects. Maintaining on an exclusion diet partially tolerant children should be considered debatable.

Mast cells are critically involved in serum-mediated vascular leakage in chronic urticaria beyond high-affinity IgE receptor stimulation.

BACKGROUND: Chronic urticaria (CU) is one of the most common skin disorders whose pathogenic mechanisms are not fully clarified. Autoimmune aetiology can be ascribed to 45% of patients with CU, and basophil histamine release is positive in 40% of cases. Our aim was to use a novel approach to evaluate the serum permeabilizing effect to identify the mediators of endothelial cell (EC) leakage and to define the role of mast cells (MCs) in the process. METHODS: Permeabilizing activity of sera from 19 patients with CU and 11 healthy blood donors was evaluated by measuring serum-induced degranulation of two MC lines, expressing (LAD2) or lacking (HMC-1) the IgE receptor. Mast cell supernatant (SN) was then incubated with an EC monolayer, and endothelial permeability was evaluated by Fluorescein isothiocyanate-bovine serum albumin leakage in a transwell system. RESULTS: All 19 patient sera failed to induce direct EC leakage, but 15/19 and 17/19 promoted degranulation of HMC-1 and LAD2, respectively. Interestingly, 85% of autologous serum skin test-negative sera were able to cause MC degranulation. Also, 17/19 SNs from HMC-1 and all SNs from LAD2 incubated with CU sera increased endothelial permeability. Endothelial cell leakage remained unchanged after Ig depletion and was prevented by antihistamine, platelet-activating factor or leukotriene antagonist. CONCLUSIONS: Our study shows that CU sera are able to degranulate MCs through an IgE- and IgG-independent mechanism. The nature of histamine-releasing factors involved is still unclear, but our finding opens new ways to the understanding of the pathogenesis of CU, particularly in patients not showing circulating autoantibodies to FcεRI or IgE.

An immunohistochemical study of leucocytes in human endometrium, first and third trimester basal decidua.

An immunohistochemical quantitative study of leucocyte subpopulations on fresh human endometrium and on biopsy specimens of first and third trimester basal decidua in normal (uncomplicated) pregnancies was performed. The most prominent population in endometrial and decidual stroma of basal decidua are macrophages. B cells as well as gamma/delta T cell receptor positive cells were found occasionally, scattered throughout the endometrial/decidual stroma. CD3+ cells were present in a relatively small number in the endometrium as well as in the first trimester basal decidua, but their number was elevated (doubled) in the third trimester of pregnancy. CD2+ cells showed a slight increase in first trimester basal decidua when compared with both endometrium and third trimester basal decidua. Cells with positive NKH-1 marker (CD56+) showed a significant increase in the first trimester, while in the third trimester their number diminished drastically. CD56:CD3 cell ratio increased to more than five times in first trimester basal decidua, while in the third trimester basal decidua decreased drastically. The mentioned increase of CD56+ cells in the first trimester and that of CD3+ cells at term suggests that these cells could have some specific function(s). However, it still has to be established whether the described quantitative changes of decidual leucocytes in basal decidua during pregnancy are of any importance for the mechanism(s) for the fetal allograft protection.

Decidual-trophoblast interactions: decidual lymphoid cell populations in basal and parietal decidua.

Immunohistochemical analysis of tissue specimens from human pregnancy decidua basalis in contact with invasive trophoblast of chorion frondosum and decidua parietalis in contact with non-invasive chorion laeve do not differ in the frequency of lymphoid cells of the following phenotypes (CD2, CD4, CD8, CD14, CD21 and gamma/delta TCR). A practical implication of this observation is that the collection of lymphoid cells from whole decidua by curettage for functional studies is justified.

Vulvar lichen sclerosus: an immunologic study.

OBJECTIVE: To investigate the seroimmunologic (CD3, CD4, CD8 lymphocytes, C3c and C4 complement fractions, and several autoantibodies) and immunohistochemical (T lymphocyte subpopulations, B lymphocytes, natural killer cells, macrophages, immunoglobulin [Ig] G, Ig M, and C3c complement fraction) characteristics of vulvar lichen sclerosus. METHODS: Serum samples from 68 women with histologically proven lichen sclerosus were compared with those from 53 healthy controls, and tissue samples from 14 of 68 women chosen at random were compared with those from 14 of 53 healthy controls. A scoring system was constructed to compare the number of cells in the tissue. RESULTS: Patients had significantly lower counts of circulating lymphocytes CD3 and CD4 than controls (P < .05) and a higher number of autoantibodies (P < .01). Analysis of the tissue samples confirmed a lower number of CD2 cells (two-tailed P = .002 in epidermis, .005 in dermis), CD3 cells (two-tailed P = .001 in epidermis and in dermis), CD4 cells (two-tailed P = .002 in epidermis, .011 in dermis), and CD8 cells (two-tailed P = .002 in epidermis, .051 in dermis) in subjects than in controls. Numbers of monocyte-macrophage cells were similar in the epidermis but different in the dermis (two-tailed P = .003). No natural killer CD56 cells or B lymphocytes (CD19-CD21) were detected in the affected areas. Deposits of IgG, IgM, and C3 were no greater in biopsy specimens of patients than in those of controls. CONCLUSION: Vulvar lichen sclerosus is not caused by a T cell-mediated response, and a viral origin is unlikely. The absence of CD19 and CD21 cells excludes local production of autoantibodies. Our data do not confirm an autoimmune pathogenesis for vulvar lichen sclerosus but help explain why systemic cortisone is of no benefit and justify the use of petroleum jelly to relieve pruritus.

Immunological patterns in monoarticular juvenile rheumatoid arthritis.

Seven pediatric patients with monoarticular arthritis, three of whom had a recent onset form and the remaining four a disease of longer duration, were examined for possible modifications of their immunological parameters. The diagnosis of JRA was made on all these patients according to the ARA criteria after a follow-up of at least two years. Humoral and cellular abnormalities of the immune system were searched for in peripheral blood, synovial fluid and synovial membrane. No evidence for complement consumption and for increased levels of immune-complexes was found in the sera and in the synovial fluids of these patients, who were all seronegative. Some patients had antinuclear antibodies in their sera and synovial fluids. With regard to the lymphocyte distribution, whereas only some patients had an increased number of circulating B cells, the majority had a decreased CD4+/CD8+ ratio in the synovial fluid compared to the ratio found in the peripheral blood. A massive infiltration of CD4+ cells and macrophages and the presence of a substantial number of OKT9+ cells was found in the synovial membranes.

Migraine: possible role of platelet insensitivity to prostaglandin E1 (PGE1).

Platelet aggregation inhibition, induced by prostaglandin E1 (PGE1), was evaluated in 38 patients affected by migraine. Our data indicate a complete insensitivity to PGE1 in these subjects. The insensitivity to PGE1 leads to decreased cyclic-AMP (cAMP) levels, determining an imbalance in the inhibitory mechanism. From this observation we can suppose that the decreased affinity of PGE1-receptors, causing decreased cAMP levels, may be involved in pathogenesis of migraine.

Immunological study of vulvar lichen sclerosus: preliminary considerations.

Sclero-atrophic lichen (LSA) is a dermatosis that is well defined from the clinical and histological viewpoints, but the etiology remains unknown. The main symptom is a permanent pruritus which results in a gynecological consultation. We have studied the immunological status of 48 patients with LSA and 33 controls. The LSA patients showed a significant diminution of peripheral CD3 and CD1 and tissue CD2, CD3, CD1 and CD8. There was no difference of IgG, IgM or tissue C3c, or serum C3c and C4. These patients also had a higher incidence of autoantibodies.

Mannose-binding lectin is produced by vaginal epithelial cells and its level in the vaginal fluid is influenced by progesterone.

Mannose-binding lectin (MBL) is a recognition molecule of the complement (C) system and binds to carbohydrate ligands present on a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL has been detected in the cervico-vaginal cavity where it can provide a first-line defence against infectious agents colonizing the lower tract of the reproductive system. Analysis of the cervico-vaginal lavage (CVL) obtained from 11 normal cycling women at different phases of the menstrual cycle revealed increased levels of MBL in the secretive phase. Part of this MBL derives from the circulation as indicated by the presence of transferrin in CVL tested as a marker of vascular and tissue permeability. The local synthesis of MBL is suggested by the finding that its level is substantially higher than that of transferrin in the secretive phase. The contribution of endometrium is negligible since the MBL level did not change before and after hysterectomy. RT-PCR and in situ RT-PCR analysis showed that the vaginal tissue, and in particular the basal layer of the epithelium, is a source of MBL which binds to the basal membrane and to cells of the outer layers of the epithelium. In conclusion, we have shown that MBL detected in CVL derives both from plasma as result of transudation and from local synthesis and its level is progesterone dependent increasing in the secretive phase of the menstrual cycle.

Mannose binding lectin and C3 act as recognition molecules for infectious agents in the vagina.

In our study we examined the early complement components in patients with bacterial vaginosis (BV), vulvovaginal candidiasis (VVC) and in healthy controls. The levels of C1q, mannose-binding lectin (MBL) and C3 were measured by ELISA in the cervicovaginal lavage (CVL) from gynaecological patients and controls. No significant differences were observed in the levels of these proteins in the three study groups. Immunofluorescence analysis of the clue cells and Candida hyphae from BV and VVC patients for surface-bound complement components showed the presence of C3, while C1q was undetectable. MBL was revealed on clue cells but not on Candida. Binding of MBL to Candida, grown or cytocentrifuged from the CVL of VVC patients, was found to be pH dependent and occurred between pH 4.5 and pH 5.5. In conclusion, we demonstrated that MBL and C3 present in the vaginal cavity act as recognition molecules for infectious agents that colonize the cervicovaginal mucosa. Our finding that MBL, but not C1q, binds to bacteria and fungi in vagina suggests that the lectin and classical pathways of complement activation may play a different role in immune defence in the female genital tract.

[Subpopulations of immunocompetent cells in the placenta in normal pregnancy and in EPH gestosis]. / Subpopulacija imunokompetentnih stanica u placenti u fizioloskim trudnocama i kod EPH gestoza.

An effort of cellular element investigations in placentas of 8 physiological pregnancies and 7 EPH gestoses is performed with the aid of specific monoclonal antibodies by the indirect immunohistochemical method. The average mean of parturient ages (primiparae and pluriparae) was 24 years in EPH gestoses. By the aid of monoclonal antibodies OKT4, OKT8, OKT11, OKT6 and OKDR, Dako-macrophage, 2H4 (Coulter) murine type, immunocompetent cells were identified in the decidua, in the stroma of chorionic villi, in the intervillous space, and in the fibrinoid masses. The semiquantitative indirect method of the immunohistochemical cellular investigation was performed by the aid of optic microscopy in 20 fields per every cutting (section) of the placental tissue. The immunocompetent active cell distribution did not reveal any special difference in the frequency of placental tissues of physiogical pregnancies and EPH gestoses, but OKT11+ cells were present in the major number in gestoses (in decidua). OKT8+ and OKDR+ cells were found in a similar number in both populations, while OKT6+ were not found at all in any investigated tissue.

Immunohistochemical detection of terminal complement complex and S protein in normal and pre-eclamptic placentae.

Terminal complement complex and S protein were searched for in term placentae obtained from 13 women with normal pregnancy and 15 patients with moderate or severe form of pre-eclampsia. Terminal complement complex was found to localize in the fibrinoid material of the decidua of the basal plate, in the stroma of the chorionic villi and in the vessel walls, as subendothelial deposits. S protein had a quite different distribution, being detected in the syncytiotrophoblast located both in the chorionic villi and in the decidua of the basal plate (DBP) and also on the endothelial cells of fetal stem vessels. Mild deposits of C3 were found in the decidua of the basal plate and also in the stroma and on the basal membranes of the villi. Reactivity for C9 neoantigen was also observed in the cytoplasm of some cells, which were recognized to be macrophages by the presence in their cytoplasm of acid phosphatase and by their reaction with a monoclonal antibody specific for macrophages. Differences in complement deposition in normal and pre-eclamptic placentae were essentially quantitative. Possible mechanisms of complement activation are discussed.

Susceptibility of human trophoblast to killing by human complement and the role of the complement regulatory proteins.

The susceptibility of trophoblast to cytolysis by human complement was investigated using cells purified to over 90% from first trimester placentae. Two assay systems were employed to measure the killing of trophoblasts, an antibody-dependent complement-mediated cytolysis and the reactive lysis. The antibody obtained from a patient with Addison's disease reacted specifically with syncytiotrophoblasts and induced a dose-dependent killing of the cells not exceeding 50% even in the presence of excess antibody and complement. The percentage of cells killed by the terminal complement complex in the reactive lysis system was somewhat higher, reaching values of 60%. Immunofluorescence analysis revealed the presence of CD46 and CD59 on all syncytiotrophoblasts, whereas CD55 was only detected on approximately 30% of the cells. Inhibition of CD46 and CD59 resulted in increased susceptibility of syncytiotrophoblasts to complement lysis. The protective function of CD55 could not be evaluated because of its reduced expression on isolated trophoblasts. These results suggest that syncytiotrophoblasts may be killed by complement and that membrane regulators to some extent protect these cells from complement damage.

Inhibition of in vitro adherence of antibody-coated red cells to monocytes by the dialyzable leukocyte extract.

The red cell-monocyte assay (RMA), which has been used to evaluate the clinical significance of red cell (RBC) antibodies, was employed to test the effect of the dialyzable leukocyte extract (DLE) on in vitro adherence to monocytes of human RBCs coated with alloantibodies or autoantibodies. The total association index (TAI) of the RMA, expressing the number of RBCs adhering to or phagocytosed by 100 monocytes, indicated a potent inhibitory activity of DLE in the test system. TAI values of 100.4 +/- 20.1 (mean +/- SD) in the control sample, consisting of RBCs coated in vitro with anti-D, dropped to 4.0 +/- 2.1 when DLE was present in the assay medium at a concentration of 0.5 U per mL. Similar results were obtained with RBCs coated with IgG antibodies in vivo. The inhibition was dose dependent and was associated with a thermolabile component of DLE. This study establishes that DLE can modulate monocyte function by inhibiting the recognition of IgG sensitized red cells.

Complement-fixing islet cell antibodies in type-1 diabetes can trigger the assembly of the terminal complement complex on human islet cells and are potentially cytotoxic.

Forty-one sera of patients with IDDM (insulin-dependent diabetes mellitus) containing complement-fixing islet cell antibodies were analyzed for their ability to activate TCC (terminal complement complex). Eighteen sera were found to promote deposition of TCC on human islets of pancreatic cryostat sections with a nonhomogeneous pattern of distribution corresponding to that of insulin. Activation of TCC by IDDM serum and binding of this complex to islet cells was confirmed using purified islets. Flow cytometric analysis of islet cell treated with a TCC+ IDDM serum showed IgG binding to the cell surface. The same serum had a cytotoxic effect on islet cells in the presence of human C. These results obtained with a homologous system of C activation by IDDM sera suggest that TCC may contribute, at least in part, to the pancreatic beta cell damage.