Immunological status in patients undergoing in vitro fertilisation: responses to hormone treatment and relationship to outcome.

We aimed to prospectively investigate the paternal antigen-induced cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to hormone treatment in women undergoing in vitro fertilisation (IVF) and to examine the predictive value of the cytokine secretion profile in the outcome of IVF treatment, in a pilot study. Twenty-five women were included and IVF treatment was successful for six and unsuccessful for 19 women. Blood samples were collected before IVF treatment, on four occasions during IVF and four weeks after embryo transfer. The numbers of Th1-, Th2- and Th17-associated cytokine-secreting cells and cytokine levels in cell supernatants were analysed by enzyme-linked immunospot-forming (ELISpot), enzyme-linked immune-sorbent (ELISA) or Luminex assay. None of the cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, TNF and GM-CSF) had any predictive value regarding IVF outcome. The majority of the cytokines reached their peak levels at ovum pick-up, suggesting an enhancing influence of the hormonal stimulation. Pregnancy was associated with a high number of IL-4-, IL-5- and IL-13-secreting cells four weeks after ET. In conclusion, the results do not support our hypothesis of a more pronounced peripheral Th1 and Th17 deviation towards paternal antigens in infertile women with an unsuccessful IVF outcome, although this is based on a small number of observations. A larger study is required to confirm this conclusion. Higher numbers of Th2-associated cytokine-secreting cells in pregnant women four weeks after ET do corroborate the hypothesis of a Th2 deviation during pregnancy.

Reduced IFN-γ and IL-10 responses to paternal antigens during and after pregnancy in allergic women.

Normal pregnancy and allergy are both characterized by a T helper (Th) 2 deviation. In the current study, we hypothesized that paternal antigen-induced cytokine responses during pregnancy would be deviated toward Th2 and an anti-inflammatory profile, and that the Th2 deviation would be more pronounced in allergic pregnant women. Blood samples were collected longitudinally on three occasions during pregnancy and two occasions post partum (pp). Of the 86 women initially included, 54 women had a normal pregnancy and completed the sampling procedures. Twelve women fulfilled the criteria for allergy (allergic symptoms and circulating immunoglobulin [Ig] E antibodies to inhalant allergens) and 20 were non-allergic (nonsensitized without symptoms). The levels of Th1- and Th2-associated cytokines and chemokines, the Th17 cytokine IL-17 and the anti-inflammatory cytokine IL-10 of the groups were compared. Paternal antigen-induced IL-4 and IL-10 responses increased between the first and the third trimester. Allergy was associated with decreased paternal antigen-induced IFN-γ and CXCL10 secretion in the nonpregnant state (one year pp) and also decreased IFN-γ/IL-4 and IFN-γ/IL-13 ratios during pregnancy. We also observed a decreased paternal antigen-induced IL-10 response in allergic compared with non-allergic women during pregnancy, along with a decreased IL-10/IL-13 ratio. In conclusion, our findings support the hypothesis of lower Th1 responses toward paternal antigens in allergic than in non-allergic women, but also indicate that allergy is associated with a lower capacity to induce anti-inflammatory IL-10 responses after paternal antigen stimulation during pregnancy.

Total and allergen-specific IgE levels during and after pregnancy in relation to maternal allergy.

Type 2 T-helper cell (Th2)-skewed immunity is associated with successful pregnancy and the ability to easily direct immune responses to a Th2-polarised profile may be an evolutionary benefit. The Th2-like immunity associated with allergic disease might generate favourable effects for the maintenance of pregnancy, but could also promote development of Th2-like immune responses and allergic disease in the offspring. The aim of this study was to explore, by using IgE as a stable proxy for Th2, the Th1/Th2 balance in allergic and non-allergic women by measuring allergen-specific and total IgE antibody levels in plasma during pregnancy and after delivery. Specific and total IgE antibody levels were determined by ImmunoCAP technology at five occasions during pregnancy (gestational weeks 10-12, 15-16, 25, 35 and 39), as well as at 2 and 12 months after delivery. Thirty-six women without and 20 women with allergic symptoms were included, of whom 13 were sensitised with allergic symptoms and 30 were non-sensitised without allergic symptoms. The levels of total IgE, but not allergen-specific IgE, were increased during early pregnancy when compared to 12 months after delivery in the sensitised women with allergic symptoms, but not in the non-sensitised women without allergic symptoms (p<0.01). This increase in total IgE levels during early pregnancy only in the sensitised women with allergic symptoms indicates that allergy is associated with an enhanced Th2 deviation during pregnancy.

Increased circulating paternal antigen-specific IFN-gamma- and IL-4-secreting cells during pregnancy in allergic and non-allergic women.

INTRODUCTION: Allergic women have been reported to give birth to more children than non-allergic women, speculatively explained by the former's predisposition for Th2 polarization, possibly favoring pregnancy. AIM: The aim of this study was to test the hypothesis that allergy is associated with more Th2-deviated responses to paternal antigens throughout pregnancy. METHODS: Blood samples were collected on six occasions during pregnancy and two occasions postpartum (pp). Of the 86 women initially included, 54 women had a normal pregnancy and completed the sampling procedures. Eleven women fulfilled the strict criteria for allergy (allergic symptoms and circulating IgE antibodies to inhalant allergens) and 23 were strictly non-allergic (non-sensitized without symptoms). The numbers of blood mononuclear cells secreting IFN-gamma and IL-4, spontaneously and in response to paternal alloantigens, were compared between the groups. RESULTS: The numbers of spontaneously as well as paternal antigen-induced IFN-gamma- and IL-4-secreting cells were similar in allergic and non-allergic pregnant women on all occasions. A similar increase in the numbers of both IFN-gamma- and IL-4-secreting cells were found in allergic and non-allergic women during pregnancy, both regarding spontaneous and paternal antigen-induced secretion. CONCLUSIONS: This study does not support the hypothesis of a more pronounced Th2-deviation to paternal antigens in allergic pregnant women compared with non-allergic pregnant women, as measured by number of cytokine-secreting cells. The observed increase of both IFN-gamma- and IL-4-secreting cells during normal pregnancy may be interpreted as a Th2-situation, since the effects of IL-4 predominate over the effects of IFN-gamma.

LRIG1 expression in colorectal cancer.

In the present study the expression of LRIG1 (leucine rich repeats and immunoglobin-like domains 1) and its relation to EGFR (epidermal growth factor receptor) was examined in tumour samples and adjacent non-neoplastic tissues from 30 patients with colorectal cancer. The LRIG1 gene, at chromosome 3p14, encodes an intergral membrane protein, which counteracts signalling by receptor tyrosine kinases belonging to the ERBB (epidermal growth factor receptor) family. LRIG1 is expressed in all tissues and organs analysed to date, including breast, brain, skin, kidney, spleen and colon. Overexpression of EGFR is seen in 70 - 90% of colorectal cancers, and is associated with a poor survival. Western blot analysis showed LRIG1 upregulation in 43% and downregulation in 43% of the colorectal cancers compared to adjacent non-neoplastic tissue. No correlation was evident between LRIG1, analysed by Western Blot and the expression of EGFR analysed by immunohistochemistry. FISH (fluoroscence in situ hybridisAtion) analysis showed increased LRIG1 copy number in one of nine tumours. Four colorectal cancer cell lines demonstrated two LRIG1 gene copies. In conclusion, there was a great heterogeneity in the expression of the LRIG1 protein in colorectal cancer, which was not related to gene dosage of the LRIG1 gene. Further studies can be of interest to evaluate whether alteration in LRIG1 expression in colorectal cancer is of biological or clinical significance.

Cytokine mapping of sera from women with preeclampsia and normal pregnancies.

INTRODUCTION: Preeclampsia is a pregnancy-specific syndrome. The immune system in preeclampsia is changed with an increased innate activity and there is a hypothesis of a shift towards Th1-type immunity. The aim of this study was to determine a spectrum of soluble immunological factors denoting different aspects of immune activation in third trimester sera from women with preeclampsia (N=15) and compare with levels in sera from normal pregnant women (N=15). MATERIAL AND METHODS: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40, IL-13, IL-15, IL-17, IFN-alpha, IFN-gamma, TNF-alpha, GM-CSF, MIP-lalpha, MIP-1beta, MCP-1, eotaxin and RANTES were measured in serum using multiplex bead arrays. The levels of soluble CD14 and soluble IL-4 receptor were measured by enzyme-linked immunoassay (ELISA). RESULTS: Preeclamptic women had significantly increased levels of circulating IL-6 (p=0.002), IL-8 (p=0.003) and soluble IL-4R (p=0.037), compared to women with normal pregnancies. CONCLUSION: This study supports the hypothesis of increased inflammatory responses in preeclampsia, illustrated by the increased levels of IL-6 and IL-8. The finding of increased levels of soluble IL-4 receptor is an intriguing finding with several interpretations, which may partly support the hypothesis of a Th1 shift in preeclampsia.

Increased copy number at 3p14 in breast cancer.

INTRODUCTION: The present study was conducted to investigate if chromosome band 3p14 is of any pathogenic significance in the malignant process of breast cancer. Genetic studies have implicated a tumour suppressor gene on chromosome arm 3p and we have proposed LRIG1 at 3p14 as a candidate tumour suppressor. The LRIG1 gene encodes an integral membrane protein that counteracts signalling by receptor tyrosine kinases belonging to the ERBB family. LRIG1 mRNA and protein are expressed in many tissues, including breast tissue. METHODS: In the present report we analysed the LRIG1 gene by fluorescence in situ hybridisation (FISH), LRIG1 mRNA by quantitative RT-PCR, and LRIG1 protein by western blot analysis. Two tumour series were analysed; one series consisted of 19 tumour samples collected between 1987 and 1995 and the other series consisted of 9 tumour samples with corresponding non-neoplastic breast tissues collected consecutively. RESULTS: The LRIG1 gene showed increased copy number in 11 out of 28 tumours (39%) and only one tumour showed a deletion at this locus. Increased LRIG1 copy number was associated with increased levels of LRIG1 mRNA (two of three tumours) and protein (four of four tumours) in the tumours compared to matched non-neoplastic breast tissue, as assessed by RT-PCR and western blot analysis. CONCLUSION: The molecular function of LRIG1 as a negative regulator of ERBB receptors questions the biological significance of increased LRIG1 copy number in breast cancer. We propose that a common, but hitherto unrecognised, breast cancer linked gene is located within an amplicon containing the LRIG1 locus at 3p14.3.

Immunology of preeclampsia.

Preeclampsia is a placenta-dependent disorder with both local and systemic anomalies with neonatal and maternal morbidity. It is manifested late in pregnancy, but the onset is during early stages of gestation. The current hypothesis regarding the aetiology of preeclampsia is focused on maladaptation of immune responses and defective trophoblast invasion. Thus, an excessive maternal inflammatory response, perhaps directed against foreign fetal antigens, results in a chain of events including shallow trophoblast invasion, defective spiral artery remodelling, placental infarction and release of pro-inflammatory cytokines and placental fragments in the systemic circulation. During normal pregnancy, trophoblasts interact in the decidua with the unique uterine NK cells, modifying their cytokine repertoire, regulating adhesion molecules and matrix metalloproteinases. The inability of trophoblasts to accomplish these changes might be a critical factor for the onset of preeclampsia. Several cytokines, produced at the maternal-fetal interface, have an impact on trophoblast invasion. It is suggested that deficiency of interleukin-10 may contribute to enhanced inflammatory responses towards the trophoblasts elicited by e.g. tumour necrosis factor-alpha and interferon-gamma. Consequently, trophoblasts subjected to a high rate of apoptosis are hampered in their invasive capacity resulting in defective transformation of spiral arteries, hypoxia, thrombosis and infarction of the placenta. The ensuing infarction of placenta leads to leakage of increasing amounts of placental fragments and cytokines in the maternal circulation and an exaggerated systemic endothelial activation as identified in preeclampsia. So far, treatment of preeclampsia is focused on signs like hypertension, whereas attempts of modifying immune responses may be a possibility in the future.

Indications of an altered immune balance in preeclampsia: a decrease in in vitro secretion of IL-5 and IL-10 from blood mononuclear cells and in blood basophil counts compared with normal pregnancy.

It has been suggested that maladaptation of the maternal immune response during pregnancy might be a causal factor for preeclampsia. This study was designed to examine the systemic immune status at both the innate level and the adaptive level in pregnancies complicated by preeclampsia (n=15) and normal pregnancies (n=15). Spontaneous and in vitro-induced secretion of IL-5, IL-6, IL-10, IL-12, IL-13 and TNF-alpha, in response to paternal blood cells and the vaccination antigens purified protein derivate of tuberculin (PPD) and tetanus toxoid (TT), was detected in cell culture supernatants from blood mononuclear cells by ELISA. Preeclamptic women showed reduced numbers of basophil granulocytes in the blood (p=0.004) and lower spontaneous secretion of IL-5 from blood mononuclear cells (p=0.016). In addition, paternal antigen-induced secretion of IL-10 was decreased in preeclampsia compared with normal pregnancy (p=0.012). No further differences between preeclampsia and normal pregnancy were found for any stimuli or cytokines. The present findings of reduced basophil numbers and lower spontaneous in vitro secretion of IL-5 in preeclampsia compared with normal pregnancy indicate a decrease in systemic Th2 immunity in preeclampsia. Furthermore, the decrease in paternal antigen-induced secretion of the immunosuppressive cytokine IL-10 in preeclampsia indicates a fetus-specific decrease in immunosuppression mediated by blood mononuclear cells. Whether these systemic changes are a cause or a consequence of preeclampsia remains to be elucidated.

Systemic Th1/Th2 cytokine responses to paternal and vaccination antigens in preeclampsia: no differences compared with normal pregnancy.

PROBLEM: A Th1-shift has been suggested to be involved in the pathogenesis of preeclampsia. This study was designed to compare Th1/Th2 related cytokine secretion in blood between women with preeclampsia (n = 15) and normal pregnancies (n = 15), using a high-sensitivity technique for cytokine detection. METHODS OF STUDY: Spontaneous as well as 'fetus-specific' and recall antigen-specific (purified protein derivate of Mycobacterium tuberculosis, tetanus toxoid and lipopolysaccharide) secretion of interferon-gamma, interleukin (IL)-4, IL-10 and IL-12 in peripheral blood mononuclear cells (PBMC) was detected by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). Fetus-specific secretion was induced by stimulation with paternal PBMC in a mixed leukocyte culture assay. RESULTS: All cytokines were secreted by PBMCs both from women with preeclampsia and women with normal pregnancies. No differences in the number of cytokine-secreting cells were found between the two groups. CONCLUSIONS: No evidence was found for a shift in the systemic Th1/Th2 responses, in preeclampsia compared with normal pregnancy. This does, however, not exclude differences in the local immune responses related to the fetoplacental unit.