Judith A. Holloway

Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry

Miniature high speed label-free cell analysis systems have yet to be developed, but have the potential to deliver fast, inexpensive and simple full blood cell analysis systems that could be used routinely in clinical practice. We demonstrate a microfluidic single cell impedance cytometer that performs a white blood cell differential count. The device consists of a microfluidic chip with micro-electrodes that measure the impedance of single cells at two frequencies. Human blood, treated with saponin/formic acid to lyse erythrocytes, flows through the device and a complete blood count is performed in a few minutes. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from CD antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Tests with patient samples showed 95% correlation against commercial (optical/Coulter) blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.

Detection of house-dust-mite allergen in amniotic fluid and umbilical-cord blood

Mononuclear cells in umbilical-cord blood display allergen-specific reactivity, but how allergen exposure occurs in utero is unknown. We investigated the presence of a common inhalant allergen (Der p 1), to which mothers are exposed throughout pregnancy, by ELISA in matched maternal blood and amniotic fluid samples at 16-17 weeks of gestation, and in matched maternal and umbilical-cord blood at term (> or =37 weeks of gestation). Der p 1 was detectable in 24 of 43 amniotic fluid samples where it was also present in maternal blood, and in 15 of 24 cord-plasma samples at significantly higher concentrations than in the maternal plasma (p=0.022). The detection of Der p 1 in the amniotic fluid and the fetal circulation provides direct evidence of transamniotic and transplacental allergen exposure.

Does atopic disease start in foetal life

The prevalence of IgE-mediated allergies, such as eczema, hay fever, and asthma, has increased dramatically over the past few decades. The short time over which this change has occurred and continues to occur indicates that the environment plays a central role in these disorders. That there is an important inherited component in these diseases is well established, and numerous genes have been identi®ed as having a possible role (1). However, a complex interplay between the environment and our genes is likely to determine disease development and progression. The environmental factors that affect the inherited susceptibility have been reviewed elsewhere (2). Here we will consider the contribution of pregnancy and foetal development in this issue.

Impaired Immunity to Intestinal Bacterial Infection in Stromelysin-1 (Matrix Metalloproteinase-3)-Deficient Mice

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55−/−, IL-12p40−/−, and IFN-γ−/− mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3−/− mice. In MMP3−/− mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3−/− mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3−/− mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3−/− mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.

Fetal exposure to intact immunoglobulin E occurs via the gastrointestinal tract

Background Consideration of the evolutionary significance of IgE might provide insight into the immunological interactions occurring in utero and during early post‐natal life that regulate later atopic disease.

Immunoregulatory molecules during pregnancy and at birth

Regulation of the maternal immune response to the fetal allograft is essential for the success of pregnancy and delivery of a well-developed neonate. Numerous mechanisms have been postulated to mediate this. We hypothesised that the potent immunosuppressive molecules TGF-beta1 and IL-10 could contribute to this regulation in the mother and neonate during gestation. In comparison to non-pregnant women, TGF-beta1 and cortisol levels were increased significantly in mid (16-18 weeks) and late pregnancy (>37 weeks, no labour), with levels of both highest in late gestation. In contrast, IL-10 levels were significantly lower in maternal plasma in mid-gestation compared with that from late pregnancy and from non-pregnant women. TGF-beta1, IL-10 and cortisol were all detectable in umbilical cord blood plasma with TGF-beta1 levels significantly decreased in association with labour in contrast to cortisol levels that increased with labour. IL-10 levels in cord plasma were comparable to those of adults and did not change with mode of delivery. Elevated levels of TGF-beta1, but not IL-10, in the maternal and neonatal circulation could have a role in immunoregulation of the maternal response to the fetal allograft as well as growth and development of the fetus.

Phenotype of fetal monocytes and B lymphocytes during the third trimester of pregnancy

The neonate typically exhibits an immature immune response compared with the adult, yet the fetus is able to generate antigen-specific responses from around 20-22 weeks of gestation. Although antigen-presenting cells (APCs) must have attained the necessary level of maturity to support this, very little is known about the phenotype and function of these populations during human fetal development. Whole blood flow cytometry was, therefore, utilised to phenotype fetal/neonatal circulating monocytes and B cells throughout the third trimester of pregnancy. The percentage of B cells (CD19+) expressing MHC Class II was comparable to the adult at all gestations, whereas the percentage of MHC Class II-positive monocytes (CD14+) increased significantly over gestation (P=0.0008) but remained lower than the adult at term. In contrast, the percentage of CD40+ or CD86+ fetal/neonatal monocytes at all gestations was comparable to the adult, but there was a maturational increase in the percentage of CD40+ or CD86+ B cells (P=0.007) to adult levels by term. The expression of CD14 itself (mean fluorescence intensity, MFI) showed a trend to increase over gestation (P=0.062) and, although all CD14+ cells expressed other receptors associated with innate immune responses (CD11b and CD35), there was fluctuation in the intensity of expression over gestation. Functional immaturity of neonatal antigen-specific immune responses could be associated with reduced co-stimulation provided by both monocytes (via reduced MHC Class II) and B cells (via reduced CD40 and CD86); altered innate responsiveness of monocytes could also contribute.

Activation of human dendritic cells by the PorA protein of Neisseria meningitidis

The major porin proteins present in the outer membrane of Neisseria meningitidis, the causative agent of life‐threatening meningitis and septicaemia, are believed to have potent immunostimulatory effects. In this study, the interactions between human monocyte‐derived dendritic cells (mo‐DC) and the PorA porin were investigated, in order to reveal the role of this protein in promoting innate and adaptive immune responses. Recombinant (r)PorA induced mo‐DC maturation, as reflected by reduced receptor‐mediated endocytosis, increased production of the chemokines IL‐8, RANTES, MIP‐1α and MIP‐1β and augmented expression of the surface markers CD40, CD54, CD80, CD86 and major histocompatibility complex class II molecules. However, rPorA induced either low level or no significant secretion of pro‐inflammatory cytokines from mo‐DC. The protein potently augmented the capacity of mo‐DC to activate both allogeneic CD4+ memory T‐cells and CD4+RA+ naïve T‐cells. In addition, rPorA appeared to inhibit the production of IL‐12p70 that follows from the interaction between CD40 on the mo‐DC and CD40‐ligand on T‐cells, thereby directing T‐cell differentiation towards a Th2 type response. These data demonstrate that PorA is involved in DC activation and in influencing the nature of the T‐helper immune response, which are important properties for generating antibody responses required for protective immunity against meningococci and for determining the immuno‐adjuvant effects of this protein.

Fetal immune responsiveness and routes of allergic sensitization

There is much interest in the role of early‐life events in the subsequent development of atopy and/or atopic disease. Despite the ongoing debate about the intrauterine exposure of the fetus to environmental allergens and the establishment of T‐cell memory, it is clear that the immunological response of the neonate at risk of atopy is more immature than that of the neonate likely to be non‐atopic. The reasons for this remain unknown, but might reflect maternally transmitted signals that adapt the neonatal immune response. An inadvertent consequence of this might be an inappropriate host response to environmental signals such as those from microbial products during early post‐natal life that result in an inability to dampen neonatal T helper 2‐skewed responses. The developing gastrointestinal tract and the exogenous factors that impact on this, such as microbial flora and breast milk, should therefore be a focus of investigation.

Phenotypic characterization of CD3–7+ cells in developing human intestine and an analysis of their ability to differentiate into T cells

We have identified a large population of CD3−7+ cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8αα homodimer. In contrast about half of CD3+ cells expressed CD4 and half expressed CD8α. A large proportion of CD3−7+ cells expressed CD56, CD94, and CD161, and whereas CD3+ T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3−7+ cells have the potential to differentiate into CD3+ cells. About half of CD3−7+ cells contain intracellular CD3ε. Rearranged TCR γ-chains were detected in highly purified CD3−7+ cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5′ Jγ segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3−7+ cells also gave rise to CD3+ T cells. Thus, we demonstrate that the CD3−7+ cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3+ T cells.