Changes In Leukocyte Populations Research Articles

Changes in Leukocyte Populations among Children Receiving a Follow‐on Cow's Milk‐Based Formula Containing Lactobacillus rhamnosus GG

BackgroundLactobacillus rhamnosus GG (LGG) is a probiotic with demonstrated health benefits, and these benefits may be due to LGG‐induced changes in the immune system.ObjectiveWe examined changes in immune cell populations and activation markers produced by the addition of LGG to a children's follow‐on formula.MethodsThis was a double blinded, randomized, controlled, parallel‐designed, prospective study. Data is reported for participants who completed the study. Participants (1–4 years of age) received either a marketed, follow‐on formula (n=8), formula supplemented with 42 mg of β‐Glucan per serving (n=10), or formula with 6 × 109 colony forming units of LGG per serving (n=9), twice a day for a 3 week period. Flow cytometry was used to immunophenotype peripheral blood leukocytes. Thirty serum cytokines were measured via multiplex analysis. Changes from baseline were analyzed by Kruksal‐Wallis. As this was a pilot study, differences with p‐values < 0.10 were considered of interest.ResultsParticipants that received LGG showed reduced levels of IL‐2 and IL‐3. They also had a reduction in the total proportion and general activation of CD4 T cells and a decrease in specific subsets of double negative T cells.ConclusionThese data suggest that LGG has an anti‐inflammatory effect by reducing available T cell populations that can cause inflammation.

In Vivo Knockdown of TAK1 Accelerates Bone Marrow Proliferation/Differentiation and Induces Systemic Inflammation

TAK1 (TGF-β Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-β genetic models, our data suggest that in our model the function of TAK1 in TGF-β signal-transduction is overruling its function in pro-inflammatory signaling.

Primary Severe Acute Respiratory Syndrome Coronavirus Infection Limits Replication but Not Lung Inflammation upon Homologous Rechallenge

Our knowledge regarding immune-protective and immunopathogenic events in severe acute respiratory syndrome coronavirus (SARS-CoV) infection is limited, and little is known about the dynamics of the immune response at the primary site of disease. Here, an African green monkey (AGM) model was used to elucidate immune mechanisms that facilitate viral clearance but may also contribute to persistent lung inflammation following SARS-CoV infection. During primary infection, SARS-CoV replicated in the AGM lung for up to 10 days. Interestingly, lung inflammation was more prevalent following viral clearance, as leukocyte numbers peaked at 14 days postinfection (dpi) and remained elevated at 28 dpi compared to those of mock-infected controls. Lung macrophages but not dendritic cells were rapidly activated, and both cell types had high activation marker expression at late infection time points. Lung proinflammatory cytokines were induced at 1 to 14 dpi, but most returned to baseline by 28 dpi except interleukin 12 (IL-12) and gamma interferon. In SARS-CoV homologous rechallenge studies, 11 of the 12 animals were free of replicating virus at day 5 after rechallenge. However, incidence and severity of lung inflammation was not reduced despite the limited viral replication upon rechallenge. Evaluating the role of antibodies in immune protection or potentiation revealed a progressive increase in anti-SARS-CoV antibodies in lung and serum that did not correlate temporally or spatially with enhanced viral replication. This study represents one of the first comprehensive analyses of lung immunity, including changes in leukocyte populations, lung-specific cytokines, and antibody responses following SARS-CoV rechallenge in AGMs.

Changes in leucocyte populations following murine bifunctional antibody infusion in colon cancer patients

This study was undertaken to determine whether infusion of a unique ZCE/CHA bifunctional antibody (BFA, 5-40 mg) could alter the composition and functions of peripheral blood leucocytes in 18 patients with colon cancer. The BFA is made by combining chemically the Fab' fragments of two murine monoclonal antibodies. One fragment (ZCE 025) binds to the carcino-embryonic antigen (CEA) and the other (CHA 225) to an epitope, present on an 111In-benzyl EDTA analog of bleomycin (BLEDTA IV) and on 111In-hydroxy-ethyl-thiourea benzyl EDTA (EOTUBE). The radiolabelled epitope (111In-BLEDTA IV or 111In-EOTUBE) was given 4 days after prelocalization with BFA. Peripheral blood samples were tested before BFA infusion, at the end of infusion (1 h later), and at 4 and 7 days post-infusion. A 50% or greater suppression in lymphocyte responsiveness to phytohaemagglutinin (PHA) and concanavalin A (Con A) was seen in 13 out of 18 and 12 out of 18 subjects, respectively, at some time after BFA infusion; this was especially evident in those patients with pre-infusion stimulation indices of greater than 50 (PHA) and/or greater than 10 (Con A). In contrast, natural killer (NK) cell cytotoxicity and oxygen radical production increased in five out of 15 and in seven out of 18 subjects, respectively. Little or no change was observed in CD3, CD4, CD8, CD16, and CD19 markers on lymphocyte subpopulations as determined by flow cytometry. These data suggest that significant changes in mitogen-induced lymphoproliferation. NK cell cytotoxicity, and oxygen radical production can occur in a substantial proportion of cancer patients after infusion of the ZCE/CHA bifunctional antibody system. The immunomodulation was unrelated to initial BFA dose, dose of BFA as a carrier, or to subsequent infusion of either form of the 111In epitope. The clinical significance of these phenomena, if any, remains to be determined.

Intraperitoneal recombinant interleukin-2 activates leukocytes in rat endometriosis

The aim of this double-blinded study was to determine changes in leukocyte populations in blood, peritoneal lavage fluid, eutopic and ectopic endometrium after treatment with recombinant rat interleukin-2 (IL-2) using an in vivo experimental model of rat endometriosis. The in vivo model involved transplanting four square fragments of autologous endometrium onto the inner surface of the abdominal wall in 20 Wistar rats. The control group was constituted by 20 sham-operated rats. Both groups were randomly treated (1-month interval treatment) with 2 intraperitoneal doses of glucose solution (5%) that did or did not contain recombinant IL-2, and animals were sacrificed 4 weeks after the last dose of treatment. Blood and peritoneal lavage were obtained during the initial and final laparotomy, whereas eutopic and ectopic endometrium were collected at the end of the experiment. Endometriotic implants were measured in each laparotomy to determine any change in size. Leukocyte populations were analyzed by flow cytometry and immunofluorescence microscopy. Cytometric results were similar in blood and peritoneal lavage. CD25+ and natural killer (NK) cell levels in peripheral blood were lower in rats with endometriosis treated with IL-2, whereas NK cells increased in lavage compared to placebo group. The percentage of macrophages and dendritic cells in blood were higher in all rats treated with IL-2, as well as peritoneal dendritic cells. Implant size of these rats decreased significantly, showing a greater number of activated lymphocytes, macrophages, NK and dendritic cells inside them. In conclusion, recombinant IL-2 induced recruitment of activated leukocytes into endometriotic-like foci, and this was related to a reduction of the implant size, suggesting potential effectiveness of IL-2 as an immunomodulatory agent in this pathology.

Leucocyte population changes in the reproductive tract of the ewe in response to insemination

Leucocyte changes after insemination may affect conceptus implantation, but information regarding leucocyte populations in the ruminant reproductive tract is limited. The present study investigated changes in leucocyte populations and distribution in the ovine reproductive tract following oestrus and insemination. Fifteen ewes were mated with a ram for 1 h and their reproductive tracts collected 3, 6, 18, 24 or 48 h later. Another 15 ewes were used as oestrus controls. Tissues were collected from 10 sites in each reproductive tract and stained with haematoxylin and eosin, Toluidine blue and immunohistochemically using a monoclonal CD68 antibody. Luminal mucus smears were collected from seven sites and stained with a modified Wright's stain and immunohistochemically. Neutrophils, eosinophils, mast cells and macrophages were identified and quantified, and temporal changes in their distribution within tissues were examined. Neutrophils and macrophages increased significantly (P < 0.05) in posterior cervical and uterine tissues following insemination. In uterine tissues, neutrophils peaked at 6 h after insemination, whereas macrophages peaked at 18-24 h. Mast cells decreased and eosinophils remained constant. Neutrophils increased significantly (P < 0.05) in the cervical and uterine lumen following insemination. In conclusion, leucocyte population changes after insemination vary between different sites in the ovine reproductive tract and may contribute to pregnancy establishment.

Changes in Leukocyte Population after Ozonated Autohemoadministration in Cows with Inflammatory Diseases

In this study, we investigated whether ozonated autohemoadministration (OAHA) influences leukocyte populations in cows with clinical inflammatory disease. Eleven cows with inflammatory disease (Inflammatory Group) and three healthy cows (Control Group) were used for this study. The CD4(+)/CD8(+) ratio in the Inflammatory Group increased significantly compared to that in the Control Group 3 to 4 days after OAHA treatment. In the Inflammatory Group, the number of CD14(+) cells decreased gradually after OAHA, but CD14(+) levels remained stable in the Control Group. The number of MHC class-II(+) cells decreased gradually in the Inflammatory Group, but increased gradually in the Control Group, and the difference between the groups was significant on day 14 after OAHA. These findings suggest a possible difference in the activation of immune response after OAHA in infected cows compared to healthy cows.

Effect of Nutritional Conditions on Changes in Leukocyte Populations in Japanese Black Calves

To clarify the effect of nutritive conditions on changes in immune cells in Japanese Black (JB) calves during the growth period, leukocyte populations were analyzed in ten healthy JB calves managed in one herd. The calves were divided into two groups: five calves in Group 1 were given insufficient nutrition, and the other five calves in Group 2 received adequate nutrition. The levels of serum total cholesterol and glucose were significantly lower in Group 1 than in Group 2 at 1 month. The numbers of CD3+, CD4+ and CD8+ cells tended to be lower in Group 1 than in Group 2 at months 1 and 2, and the difference in CD4+ was significant at month 2. The number of MHC class-II(+high) cells was significantly lower in Group 1 than in Group 2 at months 1 and 2. These results suggest that adequate nutrition might stimulate an increase in immune cells in calves during the growth period.

245. Endometrial changes in a mouse model for long-term progestin exposure: implications for break-through bleeding

A mouse model of menstruation1 was modified to evaluate the effects of long-term progestagens on the endometrium. Many women using long-acting progestin (P)-only contraceptives experience break-through bleeding (BTB) and this is the main reason for discontinuation of their use by 30% of users. To understand the mechanisms involved in BTB, ovariectomised mice treated with a strict steroid hormone regime, had etonogestrel- or levonorgestrel-releasing implants inserted subcutaneously. A decidualizing stimulus (oil) was administered into one uterine horn; decidualisation was maximal 48 h later, designated day 0 (d0). The implant was left in place for a further 45 days. Uteri were collected at 5-day intervals and analysed for morphological changes, leukocyte infiltration, and matrix metalloproteinase (MMP) expression. In uteri from mice treated with etonogestrel and examined at d0, decidualisation was maximal and very few leukocytes were present (neutrophils, NEU; Macrophages, Mac; uterine natural killer cells, uNK). Some loss of tissue integrity was seen at 5d, associated with some MMP-9 positive leukocytes. However, substantial stromal breakdown was observed at 10d and this progressed until 25d. A large infiltrate of NEU and uNKs, as well as increased MMP-9 expression, was observed at these times. At 25d there was evidence of re-epithelialisation and by 45d the tissue had fully repaired back to its pre-decidualised state. Re-epithelialisation was associated with a decline in NEU and uNKs, as well as MMP-9 positive immunostaining. The morphology of uteri from mice treated with levonorgestrel implants was compared to mice treated with etonogestrel. The changes observed were very similar with a loss of tissue integrity at 5d and tissue destruction at 10d. This mouse model mimics the changes in leukocyte populations and MMP expression observed in endometria of some women using P-only contraceptives. (1)Brasted M, White CA, Kennedy TG, Salamonsen LA. (2003) Biololgy of Reproduction 69(4), 1273–1280.

284. Leukocyte trafficking in the ovary of mice immunised with recombinant murine cyclomegalovirus expressing murine zona pellucida 3

Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (MCMV-ZP3) confers infertility characterised by depletion in ovarian tertiary follicles by day 21 post inoculation followed by a progressive depletion in primordial follicles.1 Cell mediated immune responses begin as early as day 10 post immunisation with MCMV-ZP32 with the recruitment of leukocytes before serum antibody can be clearly detected in mice. The physiological mechanisms leading to infertility in inoculated mice are being progressively delineated with the role of leukocyte subsets implicated in early pathological changes in ovarian architecture. The aim of this study was to investigate the effect of MCMV-ZP3 infection on leukocytes including T cells recruited into the ovary following infection with recombinant virus. Fifteen BALB/c female mice were randomly allocated into three groups of five animals at 6 weeks of age. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2 × 104 pfu of MCMV and MCMV-ZP3 respectively. Ovaries were retrieved at day 10, 21 and 35 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages and mAb for CD4 and CD8 positive T cells. MCMV-ZP3 inoculation increased the abundance of ovarian leukocytes including CD4 and CD8 positive T cells for all time points post immunisation except for CD8 positive T cells 21 days post infection (Table 1). These results suggest that leukocytes, including T cells, are involved in causing early changes in the ovary post infection with MCMV-ZP3 that lead to the depletion of existing ovarian follicles leading to life long infertility in mice. Further experiments are underway to investigate the role of antibody and changes in leukocyte populations in the ovary as the course of infection with recombinant virus progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control. (1)Lloyd ML, et al. (2003). Biol. Reprod. 68, 2024–32.(2)O’Leary S, et al. (2004). Reprod. Fertil. Devel. 16(Supplement), 77.

Conceptus influences the distribution of uterine leukocytes during early porcine pregnancy.

Pregnancy in humans and rodents is associated with dramatic changes in leukocyte populations within the uterus. In these species, recruitment of leukocytes, mostly natural killer (NK) lymphocytes, accompanies decidualization of endometrial stroma even in the absence of pregnancy. In the pig, a nondecidualizing species, the predominant lymphocytes in the pregnant uterus are T and/or NK cells, but their distribution relative to embryonic attachment sites has not been reported. The objective of this study was to compare the abundance of leukocytes in porcine endometrium in contact with trophoblast with that between attachment sites during the early postattachment period. Uteri were recovered on Days 15-17 (n = 4), 18 and 19 (n = 4), 21 and 22 (n = 5), and 25-27 (n = 2) of gestation and from cycling pigs during the luteal phase (Day 15; n = 3). Leukocytes were identified in uterus obtained at versus between attachment sites using an antibody reactive with all leukocytes (CD44). In all pregnant animals, leukocytes were diffusely scattered throughout the endometrial stroma but were rare or absent in the luminal epithelium. Leukocyte density was approximately 3-fold greater in endometrium in contact with conceptuses than in endometrium between attachment sites throughout the early postattachment period. Leukocyte density during the luteal phase was similar to that between attachment sites, suggesting that leukocyte recruitment was a localized response to the embryo. The ability of an individual porcine conceptus to recruit maternal leukocytes to the adjacent stroma may be a vital step in early placental development and embryo survival.

Changes in maternal peripheral leucocytes around delivery.

Changes in leucocyte populations and lymphocyte transformation with PHA were measured serially in 12 pregnant women, before and after delivery. There was a significant increase in the total numbers of leucocytes and neutrophils at delivery and for the 24 hours post partum. Lymphocyte counts increased, but not at significant levels, at or following delivery. Lymphocyte transformation rates were significantly higher before delivery and at 24 hours post partum, than at delivery or 2 hours post partum. Although plasma suppressor factors were found before and after delivery, plasma taken at delivery or 2 hours post partum was significantly more suppressive for nonpregnant lymphocytes. It is recommended that maternal blood samples should be taken before delivery for assays of maternal immunocompetence or maternofetal mixed leucocyte reactions.