Peripheral blood natural killer cells and mild thyroid abnormalities in women with reproductive failure.

Abnormalities in peripheral blood natural killer (NK) cells have been reported in women with primary infertility and recurrent spontaneous abortion (RSA) and several studies have been presented to define cutoff values for abnormal peripheral blood NK cell levels in this context. Elevated levels of NK cells were observed in infertile/RSA women in the presence of thyroid autoimmunity (TAI), while no studies have been carried out, to date, on NK cells in infertile/RSA women with non-autoimmune thyroid diseases. The contribution of this study is two-fold: (1) the evaluation of peripheral blood NK cell levels in a cohort of infertile/RSA women, in order to confirm related data from the literature; and (2) the assessment of NK cell levels in the presence of both TAI and subclinical hypothyroidism (SCH) in order to explore the possibility that the association between NK cells and thyroid function is not only restricted to TAI but also to SCH. In a retrospective study, 259 age-matched women (primary infertility [n = 49], primary RSA [n = 145], and secondary RSA [n = 65]) were evaluated for CD56+CD16+NK cells by flow cytometry. Women were stratified according to thyroid status: TAI, SCH, and without thyroid diseases (ET). Fertile women (n = 45) were used as controls. Infertile/RSA women showed higher mean NK cell levels than controls. The cutoff value determining the abnormal NK cell levels resulted ⩾15% in all the groups of women. Among the infertile/RSA women, SCH resulted the most frequently associated thyroid disorder while no difference resulted in the prevalence of TAI and ET women between patients and controls. A higher prevalence of women with NK cell levels ⩾15% was observed in infertile/RSA women with SCH when compared to TAI/ET women. According to our data, NK cell assessment could be used as a diagnostic tool in women with reproductive failure and we suggest that the possible association between NK cell levels and thyroid function can be described not only in the presence of TAI but also in the presence of non-autoimmune thyroid disorders.

Pregnancy in women with Hereditary Angioedema due to C1-inhibitor deficiency: Results from the ITACA cohort study on outcome of mothers and children with in utero exposure to plasma-derived C1-inhibitor.

Background: In women with Hereditary Angioedema (HAE) due to C1-inhibitor (C1INH) deficiency (C1INH-HAE), pregnancy counseling and treatment can be challenging. Despite the evidence of the immediate favorable outcome and safety of plasma-derived (pd)C1INH concentrate, there are no data regarding any difference among women who underwent or not pdC1INH during pregnancy or on children with in utero exposure to pdC1INH. The present interview study aimed at analyzing outcome of C1INH-HAE mothers and children according to pdC1INH-exposure during pregnancies. Methods: C1INH-HAE women who experienced at least 1 pregnancy were included from seven centers of the Italian Network for Hereditary and Acquired Angioedema (ITACA). The interview study retrospectively analyzed pregnancies who underwent (group 1) or not (group 2) pdC1INH. The overall goals of the study included immediate and long-term outcomes, in terms of outcomes in the time interval between pregnancy and survey. Results: A total of 168 pregnancies from 87 included women were analyzed. At term delivery (>37 gestation-week, GW) has been registered in 73.8% of cases, while spontaneous abortion (SA) occurred in 14.2% of cases with a mean GW 7 ± 2. The group 1 including pdC1INH-treated pregnancies comprised a third of the cohort (51/168, time interval 1.5 ± 10.4 yrs), while the group 2 represented 69.6% (117/168, time interval 32.8 ± 14 yrs). The same prevalence of SA occurred when comparing group 1 (11.7%) with group 2 (15.4%) with a similar GW at SA. The group 1 was older at the pregnancy time and younger at the interview than the group 2 (P < 0.01 for both); moreover, the group 1 showed a higher prevalence of cesarean delivery (P < 0.0001). The overall prevalence of obstetrical syndromes was similar between two groups: however, gestational diabetes was described only in pdC1INH-untreated pregnancies. In utero pdC1INH-exposed children (n = 45) did not show differences compared with unexposed ones (n = 99) in neonatal short-term outcomes. Conclusion: Through appropriate management and counseling, most of C1INH-HAE women undergo successful pregnancy and delivery. For pregnant C1INH-HAE women being treated with pdC1INH, our findings are reassuring and might lead to an improvement of both the knowledge about treatments and the experience of HAE itself.

Evidence of impaired sense of smell in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is an autosomal-dominant disorder resulting from C1-inhibitor (C1INH) deficiency. Smell impairments were found in patients affected with systemic lupus erythematosus, that, similarly to HAE, is characterized by the activation of the classical complement pathway with C4 consumption. In this study, we aimed at evaluating the sense of smell in patients with HAE. METHODS: Thirty patients with HAE and 30 healthy age- and sex-matched controls were evaluated for olfactory functions using the 3-stages Sniffin'-Sticks kit (threshold, discrimination, and identification [TDI]). TDI scores were analyzed according to complement levels (C1INH, C3, C4 and CH50), Beck depression inventory (BDI-II) and danazol treatment. RESULTS: A significant decrease in olfactory function was observed in patients affected with HAE compared with controls in total TDI score (P < 0.001), and in the discrimination (P < 0.001) and identification scores (P = 0.012). Anosmia was present only in patients with HAE (3.3%) who also exhibited more frequently hyposmia (53.3%vs 3.3%, P < 0.0001). Complement levels were reduced in patients with HAE. C4 serum levels showed positive correlation with total TDI score (P < 0.001), and with discrimination (P = 0.002) and identification (P = 0.011) scores. CH50 complement levels showed positive correlation with total TDI score (P < 0.001), and with threshold (P = 0.002) and discrimination (P = 0.011) scores. Sex, age, danazol treatment, BDI-II scores were not different between the patients and controls and did not influence TDI scores significantly. CONCLUSION: Evidence for an impaired sense of smell was found in patients with HAE. The reduction in olfactory function in these cases seems to correlate with complement C4 and CH50 levels. Immune and genetic mechanisms might play a role in this defect.

Inhibition of the complement system by glutathione: molecular mechanisms and potential therapeutic implications.

Glutathione (GSH), a component of the antioxidant defence system, plays a role in autoimmunity and the complement system is often responsible for tissue damage in autoimmune diseases. The aim of this study is to evaluate the effects of GSH on the complement system. The complement system was examined in the normal human sera (NHS) of 30 healthy subjects. Increasing quantities of GSH (1, 2, 10, 20 mg) were incubated in 1 ml of each NHS. The mixtures were evaluated for complement activities (THC, CPA and APA) and for the presence of cleavage fragments of activation of C3 and B. GSH was also incubated with human complement in the presence of classical and alternative pathway activators. The results showed an inhibitory effect of GSH on the complement system starting from a dosage of GSH≥1 mg/ml. Indeed, when NHS was incubated with GSH at such dosage, a significant reduction of the complement activities THC, CPA, and APA was observed (P<0.0001, P<0.005, P=NS, respectively), and no cleavage fragments of C3 or B were found. Further analysis demonstrated that the inhibition was exerted on C3-9 and to a lower extent on classical and alternative pathway C3-convertases. Our results indicate that GSH is capable of inhibiting the complement system. These findings are relevant for the design of interventions aimed at modulation of GSH metabolism to inhibit complement-mediated damage in autoimmune diseases.

Intravenous immunoglobulin therapy in pregnant patients affected with systemic lupus erythematosus and recurrent spontaneous abortion.

OBJECTIVES: We aimed to test the maternal and fetal outcome of SLE patients who suffered from recurrent spontaneous abortion (RSA) treated with intravenous immunoglobulin (IVIg) alone during pregnancy and whether the clinical response to IVIg treatment is accompanied by modifications of SLE-associated antibodies and of complement levels. METHODS: Twelve SLE-RSA pregnant patients were treated with high-dose IVIg and compared with 12 SLE-RSA pregnant patients treated with prednisolone and NSAIDs. They were evaluated for the clinical response [lupus activity index-pregnancy (LAI-P) scale] and for ANA, anti-dsDNA, anti Ro/SS-A or La/SS-B, aCL, LAC, C4, C3 before and during pregnancy, and before and after each treatment course. Pregnancy outcome in the two groups was also evaluated. RESULTS: The groups characteristics were homogeneous at the beginning of pregnancy. A beneficial clinical response following IVIg treatment was noted in all patients and mean LAI-P decreased from 0.72 +/- 0.43 at the beginning of pregnancy to 0.13 +/- 0.19 at the end of pregnancy (P < 0.0001). Antibodies and complement levels tended to normalize in most of the patients. These clinical and laboratory improvements were significant with respect to the control group. Pregnancy was successfully carried out in 12/12 (100%) SLE-RSA patients with a mean Apgar score of 8.92. Three patients in the control group got aborted (25%). CONCLUSIONS: IVIg has a high response rate among SLE-RSA pregnant patients and may be considered safe and effective.

Human semen inhibits T rosette formation through an opiate mediated mechanism.

Seminal plasma contains high levels of opioid peptides and both seminal plasma and endogenous opioids can influence the immune system. In order to investigate whether these two findings can be related, semen was collected from 7 normal subjects, and assayed for beta-endorphin content and for its in vitro ability to inhibit the total T rosette formation of human lymphocytes in the presence or in the absence of 10(-6) M naloxone, an universal opiate antagonist. The results were as follows: 1) immunoreactive beta-endorphin content in seminal plasma was 4 to 12 times higher than the peripheral plasma levels detected in the same subjects (76.1 +/- 42.1 SD vs 10.5 +/- 2.0 SD pg/ml); 2) increasing concentrations of seminal plasma (1%, 5%, and 10%) in RPMI 1640 significantly depressed the T rosette formation ability of lymphocytes; and 3) the simultaneous addition to the incubation mixture of 10(-6) M naloxone prevented the phenomenon, while naloxone per se was ineffective. The possibility that endogenous opioids may play a role in the immunomodulatory action of human semen is suggested.

Complement activation by cigarette smoke condensate and tobacco infusion.

The capacity of tobacco infusion and cigarette smoke condensate to activate the complement pathway was investigated in vitro by hemolytic and immunological assays. Complement activity was studied using samples of normal human serum incubated at 37 degrees C with different dilutions of tobacco infusion and cigarette smoke condensate. In all mixtures containing tobacco infusion or cigarette smoke condensate at a concentration of 0.1: 1, total hemolytic complement showed an average progressive decrease of 90% and 50%, respectively, after 60 min of incubation. This decrease was associated with a decrease of both classical and alternative pathway activities. In addition, cleavage fragments of C3, C4, and factor B were identified with immunochemical analyses. This study suggests that tobacco infusion and cigarette smoke condensate are activators of complement either through the classical or alternative pathway.

High levels of NK cells in the peripheral blood of patients affected with anti-phospholipid syndrome and recurrent spontaneous abortion: a potential new hypothesis.

OBJECTIVES: No data regarding phenotypic assets of circulating lymphocytes in anti-phospholipid syndrome (APS) are reported in the literature. Role of anti-phospholipid antibodies (aPL) in recurrent spontaneous abortion (RSA) remains uncertain, while natural killer (NK)-cells are involved in RSA pathogenesis. In this study, patients affected with APS without RSA, APS with RSA and RSA without aPL were studied for NK-cell subpopulation to evaluate its role in abortive events typical of APS. METHODS: NK-cell levels in peripheral blood of APS patients without RSA (n = 28) and in APS-RSA patients (n = 25) were evaluated by means of flow cytofluorimetry. NK-cells levels were evaluated also in RSA without aPL associated with either endocrine (n = 86), anatomic (n = 30) or idiopathic (n = 77) conditions and in 42 healthy women. RESULTS: High NK levels were found in 14/25 (56%) APS-RSA patients. Among these patients, all except one aborted before the 10th gestational week (GW), while among the remaining patients all except one aborted after the 10th GW. NK mean levels were significantly higher in APS-RSA than in all the other conditions studied, including healthy subjects, except idiopathic RSA. CONCLUSIONS: Our results demonstrate that the numbers and proportions of NK-cells are significantly higher in patients with RSA with APS than in APS without RSA. Increased numbers of NK-cells correlate with reduced gestational age at abortion in patients with APS-RSA. These data lead to a hypothesis that NK-cells contribute to the development of RSA in patients with APS. NK-cells might precipitate damage initiated by aPL or they might cause pathology in RSA independent of aPL.

Activation of human complement by crude and purified antigens from Echinococcus granulosus.

Hydatid cyst fluid of Echinococcus granulosus was found to interact with human complement. This interaction resulted in the depletion of hemolytic complement in normal serum and in the electrophoretic conversion of C3 and factor B. The results provided evidence of an activation of complement via the alternative pathway when hydatid cyst fluid was used in the mixtures. Also purified hydatid antigen preparations were found to interact with complement in vitro; this interaction resulted in the consumption of complement not only via the alternative pathway, but also via the classical pathway.

Evidence for activation of complement in patients with AIDS related complex (ARC) and/or lymphoadenopathy syndrome (LAS).

The complement system was examined in 16 patients with AIDS-related complex (ARC) (n = 5) and/or lymphoadenopathy syndrome (LAS) (n = 11). Of these patients 62.5% showed an impairment of classical and/or alternative pathway activity associated with the presence of cleavage fragments of C3 and/or B and a significant reduction of many complement factors. The data indicate pathological complement activation in these patients through the classical and/or alternative pathway. Complement activation was more severe in patients with ARC than in those with LAS, and greater in drug abusers than homosexuals. The lack of efficient complement in the patients can be considered an 'acquired complement deficiency' with possible importance in the failure to combat the HIV attack.

Mild thyroid abnormalities and recurrent spontaneous abortion: diagnostic and therapeutical approach.

PROBLEM: The aim of this study is to evaluate the role of mild thyroid abnormalities in recurrent spontaneous abortion, and to assess the effects of two different therapeutical protocols. METHOD: A prospective study in the population of recurrent aborters with mild thyroid abnormalities, evaluating the obstetric outcome in 42 patients. Sixteen thyroid autoantibodies positive patients were treated with thyroid replacement therapy, while 11 patients received intravenous immunoglobulins (IVIG). Fifteen patients, characterized by negative antithyroid antibodies, and having underlying thyroid pathology, were treated with thyroid replacement therapy. RESULTS: Among patients with thyroid antibodies, 6 out of the 11 pregnancies (54.5%) treated with IVIG ended in live birth. In the thyroid supplementation group, 13 out of 16 pregnancies (81.2%) ended in live birth. Only one pregnancy loss occurred among patients with a mild underlying thyroid pathology treated with thyroid replacement therapy. CONCLUSIONS: Mild thyroid abnormalities are associated with an increased rate of miscarriage. This poor obstetrical prognosis seems to be related to an impaired thyroid adaptation to pregnancy. Thyroid replacement therapy appears to be more effective than IVIG in preventing a new miscarriage.

Impaired human ovarian follicular fluid complement function in hereditary angioedema.

We sought and detected functionally active complement in human ovarian follicular fluid obtained during the peri-ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity, alternative pathway activity and C1 inhibitor function were present with values within the normal serum range. Active complement in follicular fluid is relevant for the function of the enzymatic multifactorial mechanism of ovulation. The presence in hereditary angioedema patients of both complement (C1 inhibitor deficiency and chronically consumed complement) and ovarian abnormalities (cystic ovaries), led us to study complement function in the follicular fluid of women of reproductive age affected with hereditary angioedema. In contrast to healthy women, hereditary angioedema patients showed dramatically reduced classical pathway activity and undetectable functional and antigenic C1 inhibitor. C4 was very low, while C3 and B were slightly reduced or within the normal serum range. This complement profile was also detected in patients' sera. Since hereditary angioedema patients often show cystic ovaries (polycystic or multifollicular), the presence of multifollicular ovaries in the two patients studied, along with complement dysfunction, may be relevant. These findings, as well as the normalisation of the ovaries found by us in hereditary angioedema patients and in the patients reported here who were undergoing danazol treatment, and the increase in C1 inhibitor and the improvement of clinical symptoms, suggest a further link between complement and ovarian function.

Complement, complement activation and anaphylatoxins in human ovarian follicular fluid.

Functionally active complement was sought and detected in human follicular fluids obtained during the pre-ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity and alternative pathway activity were present in nine fluids from four different donors with values within the normal serum range. The immunochemical analysis demonstrated the presence of complement factors from C1 to C9, of B and of C1 INH, H, I. Complement anaphylatoxins were found employing RIA techniques in amounts significantly higher than in human plasma, thus demonstrating that follicular fluid complement, at least during the pre-ovulatory period, is partially activated. A possible role for urokinase-like substances in such an activation was indicated by further in vitro experiments. The presence of active complement in follicular fluid can be relevant for the function of the enzymatic multi-factorial mechanism of ovulation.

Hereditary angioneurotic edema: clinical and laboratory findings in 58 subjects.

An in-depth analysis of clinical and laboratory findings in 58 patients affected by hereditary angioneurotic edema (HANE) is reported with special focus on problems related to the diagnosis of the disorder. The functional C1 inhibitor (C1INH) assay is the method of choice in the diagnosis of HANE, as it is capable of revealing the disorder with 100% accuracy. The immunochemical assay of C1INH detected HANE in 84.5% of the cases, i.e., immunochemical deficiency of C1INH (type I HANE). C4 was markedly reduced in both type I and type II HANE; thus, C4 levels can be particularly useful when C1INH functional tests are not available. CH50 testing is of little diagnostic value since total hemolytic complement activity is reduced in a variety of other congenital or acquired pathologies involving the complement system. The CH50 assay after incubation in low ionic strength buffer may be utilized in mass screening programs for qualitative evaluation. However, the test has the drawback of not being applicable in cases of frank hypocomplmentemia. While a depletion of the complement classical pathway was detected in most cases, no alteration in the complement alternative pathway was recorded, nor there was any reduction in immunoglobulin levels. Family history was positive in 100% of the cases. Attacks were almost always brought on by stress and/or trauma, though the causes were sometimes unknown. Edema could be cutaneous (non-pitting and non-pruritic) in 94.2%, laryngeal (often life-threatening) in 48% and abdominal (almost always painful) in 88.4% of patients. Associated pathologies were found in 2 patients, i.e., lupus rash and C3NeF-positive chronic membranoproliferative glomerulonephritis, respectively.

Cystic ovaries in women affected with hereditary angioedema.

Polycystic ovary (PCO) syndrome is biochemically characterized by abnormal gonadotropin secretion and polycystic ovaries associated with increase in size and functional activity of stromal tissue; multifollicular ovaries (MFO) are defined by the presence of multiple cysts with no increase in stromal tissue. A central (hypothalamic-pituitary) abnormality, including high plasma beta-endorphin (BE) concentrations without simultaneous elevation of ACTH, was reported for subjects with PCO syndrome. Since we have found the presence of high plasma BE concentrations in hereditary angioedema (HANE) during attacks as well as during symptom-free periods, we studied, by means of pelvic ultrasound scanning employed to determine the prevalence of PCO and of MFO, 13 women of reproductive age affected with HANE who were not on oral contraceptives. We have found PCO in 5/13 (38.4%) and MFO in 7/13 (53.8%) HANE patients. Nine patients had oligomenorrhoea (five with PCO, three with MFO, one with normal ovaries), five (three with PCO, two with MFO) were hirsute and only one (with MFO) had weight loss. No patient was obese. Mean plasma LH, testosterone, prolactin, cortisol and ACTH concentrations were normal, while FSH was significantly reduced and LH/FSH ratio increased. BE concentrations were significantly high in all the patients studied. Our results clearly demonstrate that women with HANE frequently have cystic ovaries (polycystic or multifollicular) in the presence of high BE concentrations.

Functionally active complement is present in human ovarian follicular fluid and can be activated by seminal plasma.

Human ovarian preovulatory follicular fluids (FF) from 10 women were analysed for their complement contents. Functionally active complement was detected in all the fluids studied in amounts similar to those present in normal human serum. Pooled FF was challenged by seminal plasma in order to determine whether seminal plasma could activate FF complement, the pattern of such an activation and the possible consequences on the reproductive function. FF complement activation occurred during the incubation with seminal plasma with features including alternative pathway activation, factor B and C3 conversion and reduction in total haemolytic complement, as well as an inhibition by seminal plasma of the FF complement response to a new activating challenge. Possible consequences for fertilization, implantation of a fertilized ovum and local defence mechanisms against viruses and bacteria are discussed.

C8 beta subunit deficiency in a patient with recurrent neisserial infections.

A 15-year-old woman with a history of recurrent episodes of meningococcal infections was admitted to our hospital with signs and symptoms indicating a meningeal inflammation. Since in the last few years some of the patients affected by recurrent meningococcal infections have been recognized to have selective complement deficiencies, the patient's serum was studied for determining the complement function. C8 was found to be present only in traces, with a pattern of partial identity as compared with that of the normal human serum. Moreover, total hemolytic complement was undetectable and could be completely restored with purified C8, but not with other complement components; thus, we concluded for the presence of a C8 deficiency state. Further reconstitution experiments carried out with sera having selective deficiencies of either C8 beta or C8 alpha-gamma subunits allowed us to recognize the presence of a dysfunctional C8 molecule lacking the beta chain, but possessing the alpha-gamma subunit. The clinical history of the patient, characterized by recurrent meningococcal infections, further supports the current concept of an increased susceptibility of the C8 beta-deficient patients to Neisseria meningitidis infections.

Normal fetal growth in women with antiphospholipid syndrome treated with high-dose intravenous immunoglobulin (IVIG).

Antiphospholipid antibodies are associated with fetal distress and fetal death. Although different therapeutic regimens have been used, the incidence of fetal growth retardation varies between 30 and 60 per cent of reported cases. We report the evolution of fetal growth in patients with antiphospholipid antibody syndrome treated with high-dose intravenous immunoglobulins (IVIG). Fourteen patients with a history of recurrent spontaneous abortion and immunological diagnosis of antiphospholipid syndrome were followed longitudinally. Intravenous immunoglobulin at a dose of 0.5 g/kg body weight for two consecutive days was started from the fifth week of pregnancy and repeated every 4 weeks until the 33rd week of gestation. Fetal biometry was evaluated longitudinally from the appearance of the gestational sac at 4 weekly intervals. In the period between 26 and 34 weeks, the frequency of evaluation was increased to every 14 days. Data obtained were compared with a control group of 70 fetuses with uneventful pregnancies matched for gestational age. Neonatal weight is shown in relation to the centiles for the normal population. One patient out of 14 (7.1 per cent) developed gestational hypertension and abruptio placentae. No other pregnancy complications were seen. No proteinuria was found. The mean maternal age was 31.2 +/- 3.8 years. Median birth weight was 3433 g +/- 287. The median centile of the birth weight was 65.3 +/- 18.6. Mean gestational age at delivery was 1.3 weeks. No fetal or neonatal growth retardation was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Dialysis leukopenia and hypoxemia in a patient without measurable complement activity.

We have studied complement activity, total leukocyte counts, PO2 and acid-base balance during a single hemodialysis with cuprophan membranes in a patient with hereditary angioedema and C3NeF-positive chronic membranoproliferative glomerulonephritis. Before, during and after the dialytic procedure plasma complement activity (total hemolytic complement, classical and alternative pathway activities) was not detectable and no C3-conversion occurred, while profound leukopenia (from 8,500 to 1,800 leukocytes/microliter) and hypoxemia (from 101.8 to 86 mmHg PO2) were found within 20 min from the initiation of hemodialysis. Similar results were obtained by studying the same parameters during two additional hemodialytic procedures. In vitro experiments showed that the patient's C3 could not be converted, either by cuprophan or zymosan, a specific and potent complement activator, even under optimal experimental conditions. Our data demonstrate that complement activation is not the only possible mechanism responsible for early leukopenia (as well as hypoxemia) during dialysis with cuprophan membranes.