Immunotherapy for Recurrent Pregnancy Loss: Analysis of Results From Clinical Trials

PROBLEM: Up to 80% of unexplained recurrent spontaneous abortions (RS A) are thought to have an immunologic mechanism. Yet clinical trials using immunotherapy to treat women experiencing RSA have low treatment effects. The present study was undertaken to explain the low treatment effects. METHODS: Results of clinical trials using allogeneic leukocyte immunization and intravenous (IV) immunoglobulin (Ig) are compared. The mechanisms of pregnancy loss are reviewed in light of data on frequency of karyotype abnormalities in trophoblast of failing pregnancies. RESULTS: Results of two independent analyses using allogeneic leukocyte immunization as immunotherapy for all women with RSA revealed live birth ratios of 1.16 (P = 0.03) and 1.21 (P = 0.02). When the analysis was limited to primary aborters, the live birth ratio increased to 1.46 (P = 0.006). Live birth ratio after immunotherapy for all RSA using IVIg was 1.88 (P = 0.04). Because of low treatment effects, confounders to treatment success of maternal age and number of previous abortions were studied. Chromosomal abnormalities have been identified in 55% of concepti from RSA. The frequency of chromosomal abnormalities remained constant for up to six pregnancy losses. Women with a history of primary compared to secondary RSA had a higher frequency of karyotypically abnormal concepti (χ² = 4.54, P < 0.05). Risk factors for RSA also include number of previous losses. CONCLUSION: Chromosomal abnormalities are a significant confounder when evaluating efficacy of immunotherapy for treatment of RSA. Some women with RSA have a high risk of recurrent chromosomal problems.     

Results of immunotherapy for patients with unexplained secondary recurrent abortions

Twenty-six unexplained secondary recurrent aborters underwent vaccinations by using their husband's mononuclear leukocytes according to the protocol adopted for primary recurrent aborters (group I). No mixed lymphocyte culture reaction-blocking antibodies (MLR-BAbs) detected by one-way MLR between spouses were observed in these patients prior to vaccination. In all 26 patients, MLR-BAbs appeared in their sera after vaccination with the husband's mononuclear leukocytes. Of 22 newly pregnant patients after vaccinations, pregnancy successfully continued in 20; thus, the success rate of the therapy was 90.9%. Pregnancy outcome was also analyzed in unexplained secondary recurrent aborters who revealed positive MLR-BAbs without immunotherapy (group II). In this group, in 9 of 12 patients (75.0%), the pregnancy continued successfully. The rate of continuation of pregnancy was not significantly different between groups I and II. Furthermore, the outcome of 11 pregnancies in 9 unexplained secondary recurrent aborters was analyzed in those who were negative for MLR-BAbs and had become pregnant without immunotherapy (group III). Pregnancy was successfully continued in only 2 cases (18.2%) in this group. The rate of successful pregnancy in experimental groups I and II was significantly higher compared with that in group III (P < 0.0005 and P < 0.01, respectively). Thus, immunotherapy using the husband's mononuclear leukocytes on unexplained secondary recurrent aborters with negative MLR-BAbs appears to be effective.     

Effect of oral colchicine on T cell subsets, monocytes and concanavalin A-induced suppressor cell function in asthmatic patients

Asthmatic patients have a deficiency of concanavalin A-(Con A) induced suppressor cell function. We tested whether oral colchicine 0·5 mg twice daily for 7 days could correct this immunoregulatory abnormality. Peripheral blood mononuclear cells were incubated with Con A and then suppression of proliferation was measured by co-culture of these cells with healthy volunteers’mononuclear cells and phytohaemagglutinin. Sixteen asthmatic patients had significantly (P < 0·002) decreased Con A-induced suppressor cell function (17·0±17·2%, mean ± s.d.) as compared to 13 healthy volunteers (37·9±14·9%). Oral colchicine significantly (P < 0·05) increased, though only partially corrected, these 16 asthmatic patients’Con A-induced suppressor cell function (28·1±14·3%). Asthmatic patients had an increased number of monocytes (691±289 vs 388±271/mm³ for normals, P < 0·01) and a normal number of lymphocytes, Leu 4+ total T cells, Leu 3+ helper/inducer T cells, and Leu 2+ suppressor/cytotoxic T cells as well as a normal Leu 3/Leu 2 ratio. Oral colchicine significantly (P < 0·005) decreased the number of monocytes (451±255/mm³) without significantly affecting the number of lymphocytes, Leu 4+, Leu 3+, or Leu 2+ T cells, or the Leu 3/Leu 2 ratio. These results are consistent with the hypothesis that the deficiency of Con A-induced suppressor cell function in asthmatic patients may be due, in part, to an increased number and/or abnormal activity of monocytes. If so, then oral colchicine may have partially corrected the deficiency of Con A-induced suppressor cell function by decreasing the number and/or modulating the activity of monocytes.     

Circulating NK cells and their subsets in Behçet's disease

Behçet's disease (BD) is an autoinflammatory, chronic relapsing/remitting disease of unknown aetiology with both innate and acquired immune cells implicated in disease pathogenesis. Peripheral blood natural killer (NK) cells and their CD56^Dim/CD56^Bright subsets were surface phenotyped using CD27 and CD16 surface markers in 60 BD patients compared to 60 healthy controls (HCs). Functional potential was assessed by production of interferon (IFN)‐γ, granzyme B, perforin and the expression of degranulation marker CD107a. The effects of disease activity (BD^Active versus BD^Quiet) and BD medication on NK cells were also investigated. Peripheral blood NK cells (P < 0·0001) and their constituent CD56^Dim (P < 0·0001) and CD56^Bright (P = 0·0015) subsets were depleted significantly in BD patients compared to HCs, and especially in those with active disease (BD^Active) (P < 0·0001). BD patients taking azathioprine also had significantly depleted NK cells compared to HCs (P < 0·0001). A stepwise multivariate linear regression model confirmed BD activity and azathioprine therapy as significant independent predictor variables of peripheral blood NK percentage (P < 0·001). In general, CD56^Dim cells produced more perforin (P < 0·0001) and granzyme B (P < 0·01) expressed higher CD16 levels (P < 0·0001) compared to CD56^Bright cells, confirming their increased cytotoxic potential with overall higher NK cell CD107a expression in BD compared to HCs (P < 0·01). Interestingly, IFN‐γ production and CD27 expression were not significantly different between CD56^Dim/CD56^Bright subsets. In conclusion, both BD activity and azathioprine therapy have significant independent depletive effects on the peripheral blood NK cell compartment.     

Umbilical Cord Mesenchymal Stem Cells Increase Expansion of Cord Blood Natural Killer Cells

Natural killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cell progenitors with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence, it is attractive to expand and potentially utilize these cells for adoptive immunotherapy. In this study, CB mononuclear cells were CD3-depleted by immunomagnetic microbead selection to remove T cells. This CD3^dep CB-MNC fraction was then plated for ex vivo expansion, with or without a feeder layer of irradiated umbilical cord mesenchymal stem cells (UC-MSC), with or without cytokines that have been shown to be critical for NK expansion: IL-2, IL-15, IL-3, and FLT-3L. At an average of 2 weeks of culture, there was significantly higher expansion (64.7 ± 8.4-fold) of CD56⁺/CD3⁻ NK cells in the presence of the UC-MSC feeder layer and cytokines compared to controls (no increase with feeder layer only and 6.4 ± 1.5-fold increase with cytokines only, P < .05). Contact between CD3^dep CB-MNC cells and UC-MSC augmented NK expansion. The combination of all 4 cytokines was superior to IL-2 alone or 2 cytokines combinations: mean 64.7 ± 8.4-fold expansion with 4 cytokines combination versus IL-2 alone, IL-2 + FLT-3L, IL-2 + IL-15 or IL-2 + IL-3 (12.2 ± 2.0, 14.4 ± 2.4, 10.4 ± 4.1, 25.2 ± 8.1 respectively). We also observed that only fresh CD3^dep CB-MNC preparations could be expanded reliably, whereas frozen and thawed CD3^dep CB-MNC cells did not expand consistently (mean fold increase 6.5 ± 3.2). Cytotoxicity of expanded NK cells was compared with NK cells from fresh and overnight IL-2 activated CD3^dep CB-MNC. Whereas fresh cells displayed no discernible killing, strong cytotoxicity against K562, Raji, REH, and SUP-B15 cells lines was noted after overnight activation in IL-2. Cytotoxicity of expanded NK cells against Raji, REH, and SUP-B15 was lower, which, however, correlated with a predominant expansion of CD56⁺/CD16⁻ cells known to have less cytolytic activity than CD56⁺/CD16⁺. To test the transfection efficiency in NK cells, fresh or expanded CD3^dep CB-MNC cells were electroporated with either DNA or mRNA constructs for GFP. DNA had a low transfection efficiency (<10%), whereas the one for mRNA reached 52%, but at the cost of significant cell death. Our results suggest that CB NK cell progenitors can be expanded to obtain large numbers by using an irradiated feeder of UC-MSC. They maintain an elevated cytotoxic profile, and may be genetically manipulated—all characteristics that make them suitable for cellular therapies.     

Unicompartmental and bicompartmental knee osteoarthritis show different patterns of mononuclear cell infiltration and cytokine release in the affected joints

It is still controversial which cell types are responsible for synovial inflammation in osteoarthritic (OA) joints. The aim of this study was to quantify the mononuclear cell populations and their cytokines in patients with different knee OA subtypes. Synovial membrane (SM), synovial fluid (SF) and peripheral blood (PB) were harvested from patients with unicompartmental (UC) and bicompartmental (BC) knee OA. Frequencies of mononuclear cells were assessed by flow cytometry in PB and SM. Naive SF samples were analysed for a broad variety of cytokines by multiplex analysis. SM of both groups displayed a distinct mononuclear cell infiltration, with CD14⁺ macrophages being the major cell population, followed by CD4⁺ T cells and only small numbers of CD8⁺ T, CD19⁺ B and CD16⁺CD56⁺ natural killer (NK) cells. Between the two groups, SM of BC OA showed significantly higher amounts of mononuclear cells (135·7 ± 180 versus 805 ± 675 cells/mg, P = 0·0009) and higher CD4⁺ T cell presence (3·4 ± 4·6 versus 9·1 ± 7·5%, P = 0·0267). SF of BC OA displayed significantly higher concentrations for a number of proinflammatory cytokines [CXCL1, eotaxin, interferon (IFN)‐γ, interleukin (IL)‐7, IL‐8, IL‐9, IL‐12]. UC and BC OA show significant differences in their synovial inflammatory pattern. Whereas in UC OA CD14⁺ macrophages are the predominant cell population, BC OA has a higher inflammatory profile and seems to be driven by CD14⁺ macrophages and CD4⁺ T cells. Inclusion of clinical information into the analysis of cellular and molecular results is pivotal in understanding the pathophysiology of OA.     

Immunological Effects of a Hyperinsulinaemic Euglycaemic Insulin Clamp in Healthy Males

The purpose of this study was to determine the in-vivo and in-vitro effects of insulin, at physiological and supraphysiological concentrations, on the human immune system. Ten healthy young men went through a sequential two-step hyperinsulinaemic euglycaemic clamp. Plasma insulin concentrations were increased from baseline (9.0 μU/ml) to 49.1 μU/ml after 1 h of insulin infusion (step I) and to 1281 μU/ml (step II) after 2 h of infusion. As control experiments infusions of isotonic saline were performed. The unstimulated natural killer (NK) cell activity among blood mononuclear cells (BMNC) increased in response to supraphysiological plasma insulin levels (baseline versus step II: 20.6 ± 11.3 versus 27.8 ± 14.4%). The percentages of the CD16⁺ NK cells did not change, indicating an enhanced cytotoxic capability per individual NK cell. Insulin also slightly increased the activity of NK cells in vitro. A decline at step II in the concentrations of monocytes (0.29 ± 0.09 versus 0.12 ± 0.03 × 10⁹/L), lymphocytes (1.57 ± 0.46 versus 1.22 ± 0.25 × 10⁹/L), and CD16⁺ (24.2 ± 17.5 versus 16.7 ± 11.2 × 10⁷/L), CD14⁺ (20.9 ± 10.8 versus 8.6 ± 3.9 × 10⁷/L), HLA-DR⁺ (37.2 ± 22.1 versus 19.2 ± 10.7 × 10⁷/L) and CD45RO⁺ (91.6 ± 33.4 versus 61.7 ± 6.4 × 10⁷/L) cells as well as in the percentages of CD14⁺ cells (11.2 ± 4.7 versus 6.4 ± 2.3%) and CD14⁺/HLA-DR⁺ monocytes (9.7 ± 3.9 versus 4.8 ± 2.8%) were observed. No changes were found at step I. Hyperinsulinaemia did not change the percentages of the CD3⁺, CD4⁺, CD8⁺, CD19⁺, CD56⁺, CD11a⁺, CD45RO⁺ and CD45RA⁺ cells, the numbers of circulating immunoglobulin (Ig)G-, IgA- and IgM- secreting cells, or the proliferative responses of BMNC to phytohaemagglutinin, purified derivative of tuberculin or interleukin (IL)-2. Hyperinsulinaemia did not change the in-vitro sensibility to insulin. In conclusion, supraphysiological insulin levels increased the activity of the individual NK cells, but decreased the numbers of NK cells, lymphocytes and activated monocytes. The findings are presumably of minor clinical relevance but may indicate an insulin-induced immune activation.     

Low circulating CD4(+) CD25(+) Foxp3(+) T regulatory cell levels predict miscarriage risk in newly pregnant women with a history of failure

Citation Winger EE, Reed JL. Low circulating CD4⁺ CD25⁺ Foxp3⁺ T regulatory cell levels predict miscarriage risk in newly pregnant women with a history of failure. Am J Reprod Immunol 2011; 66: 320–328 Problem The purpose of this study was to determine whether quantification of peripheral blood Treg cell levels could be used as an indicator of miscarriage risk in newly pregnant women with a history of immunologic reproductive failure. Method of Study Fifty‐four pregnant women with a history of immunologic infertility and/or pregnancy loss were retrospectively evaluated (mean age: 36.7 ± 4.9 years, 2.8 ± 2.5 previous miscarriages; 1.5 ± 1.9 previous IVF failures). Twenty‐three of these women experienced another first trimester miscarriage, and 31 of these women continued their current pregnancies past 12 weeks (‘pregnancy success’). The following immunologic parameters were assessed in the first trimester: NK cell 50:1 cytotoxicity, CD56⁺ 16⁺ CD3^? (NK), CD56⁺ CD3⁺ (NKT), TNFα/IL‐10, IFNγ/IL‐10, CD4⁺ CD25^?Foxp3⁺, total CD4⁺ Foxp3⁺ (CD4⁺ CD25⁺ Foxp3 plus CD25^? Foxp3⁺), and CD4⁺ CD25⁺ Foxp3⁺ levels. Results Patients with successful ongoing pregnancies experienced a mean (CD4⁺ CD25⁺ Foxp3⁺) ‘Treg’ level of 0.72 ± 0.52%, while those that miscarried in the first trimester experienced a mean Treg level of 0.37 ± 0.29% (P = 0.005). Markers not significantly different between the loss and success groups were NK 50:1 cytotoxicity (P = 0.63), CD56⁺ 16⁺ 3⁺ NK cells (P = 0.63), CD56⁺ 3⁺ NKT (P = 0.30), TNFα⁺IL‐10⁺(P = 0.13), IFNg⁺IL‐10⁺ (P = 0.63), and CD4⁺ 25^? Foxp3⁺ cells (P = 0.10), although total CD4⁺ Foxp3⁺ levels remained significant (P = 0.02) and CD4⁺ 25⁺ Foxp3⁺ showed the most significant difference (P = 0.005). Mean day of blood draw was 49.2 ± 36.1 days pregnant (median 39.0 days). In addition, patients with a low Treg level (<0.7%) in the first trimester experienced a significantly lower ongoing pregnancy rate than those with a higher Treg level (>0.7%) in the first trimester [44% (15/34) versus 80% (16/20); P = 0.01]. Of the 18 successful pregnancies with sequential Treg results, 85% (11/13) showed a T‐regulatory‐cell‐level increase (mean Treg change 0.33 ± 0.32), while only 40% (2/5) of the failed pregnancies showed a Treg increase (mean Treg change ?0.08 ± 0.28; P = 0.02). Conclusions From these data, we propose that CD4⁺ CD25⁺ Foxp3⁺ T regulatory cells may serve as a superior pregnancy marker for assessing miscarriage risk in newly pregnant women. Larger follow‐up studies are needed for confirmation.     

The immune system and serum glutamine during a triathlon

This study examined the influence of a triathlon on the immune system and on serum amino acid concentrations. Eight male triathletes swam 2500 m, bicycled 81 km, and ran 19 km. The concentration of total serum amino acids decreased during the race, with the lowest values occurring 2 h postexercise. Similarly, serum glutamine concentration declined from 468 (SEM 24) (prerace) to 318 (SEM 20) μmoll⁻¹ (2 h postrace) and the natural killer (NK) and lymphokine activated killer (LAK) cell activities were suppressed 2 h postexercise (P < 0.05). Blood mononuclear cell proliferation decreased during exercise with the lowest value observed after running. The leucocyte concentration increased during and after exercise due to an increase in the concentration of neutrophils and monocytes. There was no significant change in lymphocyte concentration during or after the exercise. The plasma concentration of interleukin-6 did not change and the plasma concentration of interleukin-1β and tumor necrosis factor-α were below detection limits. The LAK cell cytotoxicity, but not NK cell activity or proliferative response, was significantly correlated with serum glutamine concentrations (r = 0.39,P < 0.01). This study confirms that prolonged endurance exercise results in changes in the cytotoxic function of the NK and LAK cells as well as the proliferative response. The time-course of changes in serum glutamine concentrations were best parallelled by changes in LAK cell activities.     

EXPRESSION OF THE L1 ANTIGEN (CALPROTECTIN) BY TISSUE MACROPHAGES REFLECTS RECENT RECRUITMENT FROM PERIPHERAL BLOOD RATHER THAN UPREGULATION OF LOCAL SYNTHESIS: IMPLICATIONS FOR REJECTION DIAGNOSIS IN FORMALIN-FIXED KIDNEY SPECIMENS

The L1 antigen (calprotectin) is present in circulating monocytes but is restricted to certain subsets of tissue macrophages. Its expression is significantly increased in inflammatory bowel disease, apparently because of newly recruited monocytes. In vitro experiments were performed to substantiate lack of L1 upregulation in tissue macrophages, thereby justifying the use of this marker to detect newly recruited cells. Its reliability was further evaluated by studying mononuclear cell infiltrates characteristic of acute kidney rejection. After pro-inflammatory stimulation, monocytes matured in vitro ( n =12) as well as adherent mononuclear cells from normal small intestinal mucosa ( n =5) were examined for L1 expression by immunocytochemistry and by ELISA (cell lysates). In addition, peritubular mononuclear L1⁺ cells were examined by immunohistochemistry in routine biopsy specimens from transplanted kidneys with ( n =11) or without ( n =14) histopathologically diagnosed acute rejection. L1 was not upregulated in monocytes matured in vitro , nor in mucosal macrophages after stimulation with interferon-γ, LPS, phorbol ester, or supernatant from activated leucocytes. In transplanted kidneys with signs of acute rejection, the fraction of L1⁺ macrophages was significantly increased ( P <0·001). Because L1 is persistently downregulated in mature tissue macrophages and is formalin-resistant, it identifies young infiltrating macrophages in routinely processed biopsy material. L1 should therefore be a valuable adjunct in the diagnosis of kidney rejection.     

INCREASE IN PERIPHERAL NATURAL KILLER CELL ACTIVITY IN PATIENTS WITH AUTOIMMUNE THYROID DISEASE

Changes in the activity and number of natural killer (NK) cells in peripheral blood in patients with autoimmune thyroid disease were examined. NK activity was measured in a 4-hr ⁵¹Cr-release assay and the number of NK cells was analyzed with FITC-conjugated monoclonal antibodies by use of an automated flow cytometer. NK activity in patients with untreated Graves' disease (n=25, 39.7+13.5%, P<0.05) and Hashimoto's thyroiditis (n=18, 41.0±14.2%, P<0.05) was high compared to the activity in non-pregnant controls (n=61, 32.6±15.0%). NK activity in patients with postpartum Graves' thyrotoxicosis (n=11, 48.6±18.9%) was markedly increased compared to the activity in non-pregnant controls (P<0.01) and in postpartum controls (n=29, 33.8±15.2%, P<0.05), although the mean ages of each group did not differ significantly. Moreover, NK activities in the thyrotoxic state were significantly higher than those in the euthyroid state in the same patients with postpartum Graves' thyrotoxicosis or with postpartum destructive thyrotoxicosis. The number of CD16 positive cells increased in patients with postpartum Graves' thyrotoxicosis. However the number of CD 16 and CD57 positive cells were normal in all other groups of patients. These results indicate that an increase of NK activity is associated with exacerbation of autoimmune thyroid disease both in Hashimoto's thyroiditis and in Graves' disease and suggest that NK cells might have an important role for the control of disease activity in autoimmune thyroid disease.     

Cell-Surface CD200 May Predict Efficacy of Paternal Mononuclear Leukocyte Immunotherapy in Treatment of Human Recurrent Pregnancy Loss

Problem The allogeneic leukocytes in transfused blood can modulate the recipient’s immune system so as to induce TGF‐β‐producing suppressor cells, and the cell‐surface CD200 tolerance‐signaling molecule on mononuclear dendritic cells is required for this effect. A subset of couples with unexplained recurrent pregnancy loss appears to benefit from transfusion of allogeneic paternal blood leukocytes (LIT), and considerable effort has been devoted to characterizing those who may benefit. Some data has been accumulated for LIT as sole therapy in patients with classical spontaneous abortions with respect to dose–response, duration of protection, need for boosting, excluding patients with autoimmunity, and inefficacy of paternal mononuclear cells stored at 4°C overnight before use which causes loss of cell‐surface CD200. Recent data emphasize an important role of expression of the CD200 tolerance‐signaling molecule on cells used to prevent abortions both in mice and humans. Method of study An observational study of outcome as a function of the number of CD200⁺ paternal mononuclear cells was performed. Fourteen patients constituted the pilot group. Patients with autoimmunity who had failed inspite of treatment with IVIG + Heparin + Aspirin ± Prednisone were allowed to have paternal mononuclear cells added to their therapy. CD200 on purified paternal blood mononuclear cells was measured by flow cytometry. Results The number of CD200⁺ cells administered was significantly greater in women achieving pregnancy (39.2 × 10⁶ versus 20.8 × 10⁶, P < 0.025) and in those who achieved a live birth (50.2 × 10⁶ versus 20.8 × 10⁶, P < 0.005) compared to those who did not achieve pregnancy, and % of paternal cells that were CD200⁺ was greater (11–12.5% versus 5.6%, P < 0.01). Amongst those achieving pregnancy which failed, the CD200⁺ cell dose was not significantly different from the non‐pregnant group (30.5 × 10⁶ versus 20.8 × 10⁶). Conclusion The number of CD200⁺ paternal mononuclear leukocytes may be an important determinant of subsequent reproductive outcome in a subset of patients. A lower % CD200⁺ cell number may also reflect hitherto unappreciated paternal factors bearing on reproductive success. It is feasible to recruit women to enter observational studies and to obtain useful data as a foundation for further studies. More complete patient characterization in a larger study is needed.     

Natural killer cell activity and cytokine production after in vitro immunoglobulin treatment of lymphocytes derived from pregnant women with or without risk for spontaneous abortion

OBJECTIVE: To investigate the possible mechanism of action effective in immunoglobulin G (IgG) treatment of recurrent spontaneous abortion (RSA). The effect in vitro of a commercially available intravenous immunoglobulin (IvIg) on the rate of interleukin (IL)-10 and IL-12 positive cells (Th1/Th2 balance) and on natural killer (NK) cell activity in populations of peripheral lymphocytes of healthy pregnant women and women at risk for premature pregnancy termination was studied. Primary habitual aborters as well as women showing clinical symptoms (bleeding or regular uterine contractions) of threatened premature pregnancy termination were included. METHODS: Lymphocytes of 20 pregnant women were tested. Five different batches of an IvIg with reported immunomodulatory potential were used at a concentration of 10 mg/mL. Cytokine profiles of the lymphocytes were determined by immunocytochemistry. For testing of NK cell activity, the 4 hr single cell cytotoxicity assay was used. RESULTS: Incubation with IgG of lymphocytes from recurrent spontaneous aborters concomitantly and significantly decreased the rate of IL-12 positive cells (P < 0.01) and increased the rate of IL-10 positive cells (P < 0.01), whereas such treatment had no significant effect on lymphocytes of pregnant women not at risk of abortion. Dialysis or heat treatment (56 degrees C, 30 min) of the IgG preparations did not modify the effect. Elevated NK cell activity of women at risk for premature pregnancy termination significantly decreased after IgG incubation of cells in all cases, whereas NK cell activity of normal pregnancy lymphocytes was not altered. CONCLUSION: This study suggests that incubation of peripheral lymphocytes from RSA patients with polyclonal polyspecific IgG alters cytokine profiles and NK activity while the same treatment does not affect lymphocytes of healthy pregnant women. These data might add to the understanding of mechanisms of action of IvIg in prevention of recurrent pregnancy loss.     

Effects of Ganopoly® (A Ganoderma lucidum Polysaccharide Extract) on the Immune Functions in Advanced‐Stage Cancer Patients

Preclinical studies have established that the Ganoderma lucidum polysaccharide (GLPS) fractions have potent anti‐tumor activity, which has been associated with the immuno‐stimulating effects of GLPS. However, it is unclear whether GLPS has immuno‐modulating effects in humans in vivo. This study aimed to investigate the effects of Ganopoly^®, the polysaccharides fractions extracted from G. lucidum, on the immune function of advanced‐stage cancer patients. Thirty‐four advance‐stage cancer patients were entered onto this study, and treated with 1800 mg Ganopoly^®, three times daily orally before meals for 12 weeks. Immune parameters (cytokines, T cell subsets, mitotic response to phytohemagglutinin (PHA) and natural killer (NK) activity) were compared between baseline and after 12‐week treatment. Thirty patients are assessable for their immune functions. Treatment of Ganopoly^® for 12 weeks resulted in a significant (P < 0.05) increase in the mean plasma concentrations of interleukin (IL‐2), IL‐6, and interferon (IFN)‐γ, whereas the levels of IL‐1 and tumor necrosis factor (TNF‐α) were significantly (P < 0.05) decreased. A marked variability among patients with advanced‐stage cancer was observed in the numbers of each lymphocyte subset at baseline. The mean absolute number of CD56⁺ cells was significantly (P < 0.05) increased after 12‐week treatment of Ganopoly^®, whereas the numbers of CD3⁺, CD4⁺, and CD8⁺ were just marginally increased compared to baseline levels, with the CD4:CD8 T cell ratios unchanged. PHA responses after 12‐week treatment with Ganopoly^® were enhanced in most patients, when compared to pretreatment baselines (P < 0.05). In addition, Ganopoly® treatment resulted in a significant increase (P < 0.05) in the mean NK activity compared to baselines (34.5 ± 11.8% vs 26.6 ± 8.3%). The present study indicates that Ganopoly^® enhanced the immune responses in patients with advanced‐stage cancer. Clinical evaluations of response and toxicity are ongoing.     

Enhancement of the Expression of Progesterone Receptor on Progesterone-Treated Lymphocytes After Immunotherapy in Unexplained Recurrent Spontaneous Abortion

PROBLEM: The immunological mechanism of an effective immunotherapy with paternal lymphocytes for unexplained recurrent spontaneous abortion (RSA) is not yet clear. Previous studies revealed that progesterone plays an important role in maintaining normal pregnancy and lower expression of progesterone receptor (PGR) on lymphocytes was found in RSA. Therefore, it was of interest to investigate whether immunotherapy for RSA would be able to enhance the expression of PGR on lymphocytes of RSA. METHOD: PGR expression on lymphocytes was analyzed with indirect immunofluorescence using flow cytometry. RESULTS: There was no change of PGR expression on PBL of RSA between pre- and post-immunotherapy (P > 0.05), while in the presence of 10.0 ug/ml progesterone for 24 h, PGR expressed on PBL on post-immunotherapy was increased significantly as compared with that of pre-immunotherapy in successful cases (P < 0.05) and decreased in abortive cases (P < 0.05). Most PGR was expressed on both CD4⁺ and CD8⁺ lymphocyte subsets. In successful cases, CD8⁺PGR⁺ subset of post-immunotherapy was found to be increased significantly (P < 0.05) in comparison with that of pre-immunotherapy. CONCLUSION: The data in the present study suggest that immunotherapy for RSA induced a higher expression of PGR on progesterone-treated lymphocytes, which may be involved in successful pregnancy.     

Modulation of NK Cell Lytic Function by Endometrial Secretory Factors: Potential Role in Endometriosis

PROBLEM: Development and progression of endometriosis have been suggested to be related to a defect of NK cells in the ability to eliminate endometrial fragments regurgitated with menstrual debris in ectopic sites. However, it has not been clarified yet whether the origin of this defect has to be attributed to an intrinsic alteration of NK cell population or to a consequence of the environmental system. The present study was designed to evaluate whether soluble factors of endometrial origin might directly interfere with NK cell lytic function. METHOD: Media conditioned by 20 endometrial stromal cultures (10 from endometriosis patients and 10 from women without the disease) were examined to test their effects on NK cell-mediated cytotoxicity directed against endometrial stromal targets. NK cells were purified from peripheral blood samples by a two steps panning technique and incubated overnight with and without endometrial supernatants. Thereafter, the percentage of lysis against endometrial cells was detected in a ⁵¹Cr cytotoxicity assay. RESULTS: Media conditioned by the overall endometrial stromal cultures exerted a significant suppressive effect (P < 0.001) on NK cell function when compared to control NK fractions (mean percentage of cytotoxicity ± SEM 17.46 ± 2.8 and 29.41 ± 1.49, respectively). No inhibitory effect was detect with conditioned media obtained from peritoneal cells and keratinocytes. Moreover, NK cell-mediated cytotoxicity resulted significantly more inhibited by the addition of endometrial supernatants obtained from endometriosis patients (mean percentage cytotoxicity ± SEM 13.55 ± 2.3, P < 0.05) when compared to those from women without the disease (mean percentage cytotoxicity ± SEM 21.38 ± 4.9). CONCLUSION: The results of this study indicate the existence of soluble non-specific factor(s) released by human endometrial cells able to interfere with NK cell killing. These factors could participate in the local regulation of the immunological mechanisms suggested to be involved in the development of endometriosis.     

Ro52/TRIM21‐deficient expression and function in different subsets of peripheral blood mononuclear cells is associated with a proinflammatory cytokine response in patients with idiopathic inflammatory myopathies

The presence of anti‐Ro52/tripartite motif 21 (Trim21) autoantibodies has been associated with a distinctive clinical profile and has gained value as a prognostic marker in idiopathic inflammatory myopathies (IIM). The aim of the present work was to analyse Ro52/Trim21 expression in different subsets of peripheral blood mononuclear cells (PBMCs) of patients with IIM, as well as the ubiquitination profile and its association with proinflammatory cytokine production. We included 18 patients with recent‐onset IIM and 18 age‐ and gender‐matched healthy donors. PBMCs were isolated and different subsets (CD4⁺, CD8⁺, CD14⁺) were purified by magnetic selection. The expression of Ro52/Trim21 in different PBMC subsets of patients with IIM and healthy donors was analysed by Western blot. We assessed the presence of myositis‐specific and associated autoantibodies by enzyme‐linked immunosorbent assay (ELISA). Cytokine levels were measured by cytometric bead array. Patients with IIM showed decreased protein expression of Ro52/Trim21 in comparison to healthy controls in PBMC (0·97 ± 0·60 versus 1·84 ± 0·92, P = 0·016), CD4⁺ lymphocytes (0·79 ± 0·54 versus 2·41 ± 0·78, P = 0·017), and monocytes (0·87 ± 0·35 versus 1·89 ± 0·20, P < 0·001). There were no significant differences among IIM groups. Also, a lower K48‐mediated ubiquitination profile was found, predominantly in CD4⁺ lymphocytes. Furthermore, after mitogenic stimulation, there was a higher synthesis of proinflammatory cytokines by T cells [interleukin (IL)‐17A and tumour necrosis factor (TNF)‐α] and monocytes [IL‐6 and interferon (IFN)‐α] from IIM patients compared with healthy controls. Our data suggest that patients with IIM, mainly DM, are characterized by a deficient expression of Ro52/TRIM21 in different PBMC subsets (CD4⁺ lymphocytes and monocytes), along with lower K48‐mediated ubiquitination, which is associated with a proinflammatory cytokine response.     

Th1 and Th2 cytokine profiles in recurrent aborters with successful pregnancy and with subsequent abortions

BACKGROUND: This study compared Th1-Th2 cytokine profiles in a subgroup of recurrent aborters who had an abortion with those in a subgroup of recurrent aborters who had a successful pregnancy. METHODS: Fifty-four women with a history of at least three normal pregnancies, 24 women with a history of recurrent spontaneous abortion (RSA) followed by abortion (RSA-->A) and 39 women with a history of RSA followed by normal pregnancy (RSA-->N) were studied. Blood samples and placentas were obtained at the time of delivery or abortion; peripheral blood mononuclear cells were stimulated separately with phytohaemagglutinin and with autologous placental cells, and the secreted cytokines estimated. RESULTS: Peripheral blood mononuclear cells from the RSA-->N subgroup secreted higher concentrations of Th1-type cytokines as compared with normal pregnant women, indicating a higher Th1 bias in these women. However, women in the RSA-->N subgroup had significantly higher concentrations of Th2 cytokines as compared with women in the RSA-->A subgroup. A comparison of Th1:Th2 cytokine ratios indicated a higher Th2 bias in RSA-->N women as compared with RSA-->A women. CONCLUSIONS: We conclude that abortion-prone women who proceed to have successful pregnancy are more Th2-biased than abortion-prone women who abort, and that recurrent aborters who undergo spontaneous abortion have a stronger Th1 bias than aborters who have normal pregnancy.     

Sensitivity of somatic mutations in human umbilical cord blood to maternal environments

To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (M_f), and glycophorin A (GPA) variant erythrocytes (V_f) of both allele-loss (ø/N) and allele-loss-and-duplication (N/N) phenotypes in umbilical cord blood. The mean hprt M_f (1.40 ± 1.11 × 10^?6, N = 66) and GPA V_f (ø/N 4.0 ± 2.2 × 10^?6, N = 114; N/N 2.7 ± 2.0 × 10^?6, N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt M_f was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 ± 0.41 × 10^?6, N = 45, P < 0.01; t test, P < 0.01, Mann-Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt M_f (0.70 ± 0.49 × 10^?6) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann-Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated M_f revealed a significant decrease in the relative contribution of gross structural mutations to the overall M_f (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, x² test), suggesting that the higher M_f resulted from an elevated level of “point” mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt M_f or GPA V_f. © 1995 Wiley-Liss, Inc.