Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

Background aimsKey obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells.MethodsWe delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3⁺Vα24⁺Vβ11⁺ iNKT cells.ResultsGene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4⁺ and CD8⁺ responder T cells.DiscussionThese data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.     

Potential Role of IL-17-Producing iNKT Cells in Type 1 Diabetes

We explored in this study the status and potential role of IL-17-producing iNKT cells (iNKT17) in type 1 diabetes (T1D) by analyzing these cells in patients with T1D, and in NOD mice, a mouse model for T1D. Our analysis in mice showed an increase of iNKT17 cells in NOD vs control C57BL/6 mice, partly due to a better survival of these cells in the periphery. We also found a higher frequency of these cells in autoimmune-targeted organs with the occurrence of diabetes, suggesting their implication in the disease development. In humans, though absent in fresh PMBCs, iNKT17 cells are detected in vitro with a higher frequency in T1D patients compared to control subjects in the presence of the proinflammatory cytokine IL-1β, known to contribute to diabetes occurrence. These IL-1β-stimulated iNKT cells from T1D patients keep their potential to produce IFN-γ, a cytokine that drives islet β-cell destruction, but not IL-4, with a reverse picture observed in healthy volunteers. On the whole, our results argue in favour of a potential role of IL-17-producing iNKT cells in T1D and suggest that inflammation in T1D patients could induce a Th1/Th17 cytokine secretion profile in iNKT cells promoting disease development.     

SNP/haplotype associations in cytokine and cytokine receptor genes and immunity to rubella vaccine

An effective immune response to vaccination is, in part, a complex interaction of alleles of multiple genes regulating cytokine networks. We conducted a genotyping study of Th1/Th2/inflammatory cytokines/cytokine receptors in healthy children (n = 738, 11–19 years) to determine associations between individual single-nucleotide polymorphisms (SNPs)/haplotypes and immune outcomes after two doses of rubella vaccine. SNPs (n = 501) were selected using the ldSelect-approach and genotyped using Illumina GoldenGate™ and TaqMan assays. Rubella-IgG levels were measured by immunoassay and secreted cytokines by ELISA. Linear regression and post hoc haplotype analyses were used to determine associations between single SNPs/haplotypes and immune outcomes. Increased carriage of minor alleles for the promoter SNPs (rs2844482 and rs2857708) of the TNFA gene were associated with dose-related increases in rubella antibodies. IL-6 secretion was co-directionally associated (p ≤ 0.01) with five intronic SNPs in the TNFRSF1B gene in an allele dose-related manner, while five promoter/intronic SNPs in the IL12B gene were associated with variations in IL-6 secretion. TNFA haplotype AAACGGGGC (t-statistic = 3.32) and IL12B promoter haplotype TAG (t-statistic = 2.66) were associated with higher levels of (p ≤ 0.01) rubella-IgG and IL-6 secretion, respectively. We identified individual SNPs/haplotypes in TNFA/TNFRSF1B and IL12B genes that appear to modulate immunity to rubella vaccination. Identification of such “genetic fingerprints” may predict the outcome of vaccine response and inform new vaccine strategies.     

A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer

HLA-mismatched natural killer (NK) cells have shown efficacy in acute myeloid leukemia, and their adoptive transfer in patients with other malignancies has been proven safe. This phase I clinical trial was designed to evaluate safety (primary endpoint) and possible clinical efficacy (secondary endpoint) of repetitive administrations of allogeneic, in vitro activated and expanded NK cells along with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). Patients with unresectable, locally advanced/metastatic NSCLC receiving 1st/2nd line chemotherapy were eligible to receive 2–4 doses of activated NK cells from two relative donors. Donor’s CD56⁺ cells were cultured for 20–23 days with interleukin-15 (IL-15) and hydrocortisone (HC) and administered intravenously between chemotherapy cycles. Premedication with corticosteroids and/or H1 inhibitors was allowed. Sixteen patients (performance status 0–1) with adenocarcinoma (n = 13) or squamous cell carcinoma (n = 3) at stage IIIb (n = 5) or IV (n = 11) receiving 1st (n = 13) or 2nd (n = 3) line treatment were enrolled. Fifteen patients received 2–4 doses of allogeneic activated NK cells (0.2–29 × 10⁶/kg/dose, median 4.15 × 10⁶/kg/dose). No side effects (local or systemic) were observed. At a median 22-month follow-up (range, 16.5–26 months) 2 patients with partial response and 6 patients with disease stabilization were recorded. Median progression free survival and overall survival were 5.5 and 15 months, respectively. A 56% 1-year survival and a 19% 2-year survival were recorded. In conclusion, repetitive infusions of allogeneic, in vitro activated and expanded with IL-15/HC NK cells, in combination with chemotherapy are safe and potentially clinically effective.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

Maternal and cord plasma cytokine and chemokine profile in pregnancies complicated by asthma

The mechanisms contributing to worsening of asthma during pregnancy have not been well characterized. Both asthma and pregnancy are conditions associated with a skewing of the immune response from T helper (Th) 1 toward a Th2 response. We hypothesise that worsening of asthma during pregnancy may be due to an enhanced production of circulating proinflammatory cytokines and chemokines and this may be modified by the use of inhaled glucocorticoid treatment. Peripheral blood was collected from asthmatic (n = 35) and control non-asthmatic patients (n = 13) in the third trimester (30–37 weeks) of pregnancy. Fetal blood was collected from the umbilical vein of the placenta after delivery from normal (n = 24) and pregnancies complicated by asthma (n = 24). Plasma samples were assayed for IL-6, -8, eotaxin and RANTES using conventional ELISA. In addition, a range of Th1 and Th2 cytokines measured using Luminex system. There were no significant differences in the levels of maternal IL-6, IL-8, eotaxin and RANTES between asthmatics and nonasthmatics. The results of this study suggest that the presence of asthma does not result in an enhanced circulation of Th2 related cytokines and chemokines during the third trimester of pregnancy. Furthermore peripheral blood cytokine concentrations appear unaffected by inhaled glucocorticoid treatment. Cord plasma eotaxin concentrations were increased in pregnancies complicated by asthma, compared with control. This is the first study to show increased eotaxin production in the feto-placental unit of asthmatic pregnancies and may be one mechanism by which allergy susceptibility is increased in the offspring of asthmatic women.     

SNPs in the bovine IL-10 receptor are associated with somatic cell score in Canadian dairy bulls

Altering the balance between pro- and anti-inflammatory responses can influence an animal’s susceptibility to acute or chronic inflammatory disease; bovine mastitis is no exception. Genetic variation in the form of single nucleotide polymorphisms (SNPs) may alter the function and expression of genes that regulate inflammation, making them important candidates for defining an animal’s risk of developing acute or chronic mastitis. The objective of the present study was to identify SNPs in genes that regulate anti-inflammatory responses and test their association with estimated breeding values (EBVs) for somatic cell score (SCS), a trait highly correlated with the incidence of mastitis. These genes included bovine interleukin-10 (IL-10) and its receptor (IL-10R), and transforming growth factor β1 (TGF-β1) and its receptor (TGF-βR). Sequencing-pooled DNA allowed for the identification of SNPs in IL-10 (n = 2), IL-10Rα (n = 6) and β (n = 2), and TGF-β1 (n = 1). These SNPs were subsequently genotyped in a cohort of Holstein (n = 500), Jersey (n = 83), and Guernsey (n = 50) bulls. Linear regression analysis identified significant SNP effects for IL-10Rα 1185C>T with SCS. Haplotype IL-10Rα AAT showed a significant effect on increasing SCS compared to the most common haplotype. The results presented here indicate that SNPs in IL-10Rα may contribute to variation in the SCS of dairy cattle. Although functional studies are necessary to ascertain whether these SNPs are causal polymorphisms or merely in linkage with the true causal SNP(s), a selection program incorporating these markers could have a beneficial influence on the average SCS and productivity of a dairy herd.     

Haplotypes of IL-10 promoter variants are associated with susceptibility to severe malarial anemia and functional changes in IL-10 production

Plasmodium falciparum malaria is one of the leading global causes of morbidity and mortality with African children bearing the highest disease burden. Among the various severe disease sequelae common to falciparum malaria, severe malarial anemia (SMA) in pediatric populations accounts for the greatest degree of mortality. Although the patho-physiological basis of SMA remains unclear, dysregulation in inflammatory mediators, such as interleukin (IL)-10, appear to play an important role in determining disease outcomes. Since polymorphic variability in innate immune response genes conditions susceptibility to malaria, the relationship between common IL-10 promoter variants (−1,082A/G, −819T/C, and −592A/C), SMA (Hb < 6.0 g/dL), and circulating inflammatory mediator levels (i.e., IL-10, TNF-α, IL-6 and IL-12) were investigated in parasitemic Kenyan children (n = 375) in a holoendemic P. falciparum transmission area. Multivariate logistic regression analyses demonstrated that the −1,082G/−819C/−592C (GCC) haplotype was associated with protection against SMA (OR; 0.68, 95% CI, 0.43–1.05; P = 0.044) and increased IL-10 production (P = 0.029). Although none of the other haplotypes were significantly associated with susceptibility to SMA, individuals with the −1,082A/−819T/−592A (ATA) haplotype had an increased risk of SMA and reduced circulating IL-10 levels (P = 0.042). Additional results revealed that the IL-10:TNF-α ratio was higher in the GCC group (P = 0.024) and lower in individuals with the ATA haplotype (P = 0.034), while the IL-10:IL-12 ratio was higher in ATA haplotype (P = 0.006). Results presented here demonstrate that common IL-10 promoter haplotypes condition susceptibility to SMA and functional changes in circulating IL-10, TNF-α, and IL-12 levels in children with falciparum malaria. The study was approved by the ethical and scientific review committees at the Kenya Medical Research Institute and the institutional review board at the University of Pittsburgh.     

IL-1β (−511T/C) gene polymorphism not IL-1β (+3953T/C) and LT-α (+252A/G) gene variants confers susceptibility to visceral leishmaniasis

Lymphotoxin-α (LT-α) and interleukin-1beta (IL-1β) are proinflammatory cytokines playing important roles in immunity against Leishmania infection and the outcome of the disease. As cytokine productions are under the genetic control, this study tried to find any probable relationship between these cytokine gene polymorphisms and the susceptibility to visceral leishmaniasis in Iranian pediatric patients. Ninety-five pediatric patients involved with visceral leishmaniasis and 128 non-relative healthy people, from the same area as the patients, were genotyped for LT-α (+252A/G) and IL-1β (+3953T/C and −511T/C) gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). There was not found any significant differences in allele and genotype frequencies of LT-α (+252A/G) and IL-1β (+3953) among the study groups. However, the frequency of IL-1β −511TT genotype was higher in the controls (P = 0.0004) while the frequency of IL-1β −511CC genotype and C allele were higher in the patients (P = 0.008 and P = 0.00006, respectively). Furthermore, IL-1β CC (−511/+3953) haplotype was more frequent in VL patients compared with the controls (P = 0.0002) and the distribution of TT haplotype was higher in the controls compared with the patients (P = 0.003). In conclusion, based on the results, IL-1β −511C allele, CC genotype and CC (−511/+3953) haplotype could be considered as the susceptibility factors for visceral leishmaniasis while IL-1β −511TT genotype, T allele and TT haplotype (−511/+3953) might be counted as the influential factors for resistance to the disease.     

Mice Immunization with Radioattenuated Yeast Cells of <Emphasis Type="Italic">Paracoccidiodes brasiliensis</Emphasis>: Influence of the Number of Immunizations

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-γ were produced in mice of Group 1. In Group 2, only IFN-γ was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.     

−509C>T polymorphism in the TGF-β1 gene promoter,impact on the hepatocellular carcinoma risk in Chinese patients with chronic hepatitis B virus infection

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The risk for developing HCC increases with severity of inflammation and fibrosis. Transforming growth factor-β1 (TGF-β1) is most frequently upregulated in tumor cells. The most studied −509C>T polymorphism of TGF-β1 gene has been associated with colorectal, gynecologic, and lung cancers. To assess whether this polymorphism in TGF-β1 gene is associated with susceptibility to and/or clinicopathologic characteristics of HBV-related HCC, a total of 575 patients with chronic HBV infection and 299 healthy volunteers with no evidence of recent or remote HBV infection were prospectively enrolled. The patients were divided into two groups: those without (n = 196) and those with HCC (n = 379). These 379 HCC patients with chronic HBV infection were designated as cases, the remaining 196 patients without HCC and 299 healthy volunteers served as disease and healthy controls, respectively. −509C>T polymorphism in the TGF-β1 gene promoter was studied using restriction fragment-length polymorphism. In addition, tumor tissues of liver (n = 60) were obtained from the studied HCC patients for measurement of TGF-β1 mRNA expression levels. We also assessed the plasma TGF-β1 levels of HBV patients without (n = 94) or with HCC (n = 136) and healthy subjects (n = 120). In our study group, the risk of HCC in Chinese patients with HBV infection was significantly lower with the TT genotypes than in those with the CC genotypes at position −509 of TGF-β1 gene (P = 0.01). In addition, in the case group, patients with the CC genotype had a statistically significant higher median plasma TGF-β1 or liver tumor tissue TGF-β1 mRNA level compared with the individuals with the TT genotype. However, in a subsequent analysis of the association between this polymorphism and clinicopathological characteristics including tumor number, size, grade, stage, and invasiveness, there was no significant difference in both the distribution of genotype or allelic frequency within HCC patients, indicating that −509C>T exchange in TGF-β1 gene may play an important role in the occurrence, not the progression of HBV-related HCC through influencing plasma concentrations of TGF-β1 or TGF-β1 mRNA expression of liver tumor tissue.     

Calcitriol attenuates TLR2/IL-33 signaling pathway to repress Th9 cell differentiation and potentially limits the pathophysiology of rheumatoid arthritis

There is limited information regarding the TLR2 signaling pathway involved in Th9 cell differentiation. The role of calcitriol in regulating TLR2-mediated Th9 cell development is unknown. Thus, we aimed to unravel the TLR2 signaling pathway in Th9 cells and its regulation by calcitriol. We have used n?=?5–6 animals for each murine experiment. Human studies involved five healthy volunteers. Moreover, ten healthy individuals and ten RA patients were included in the study. Murine and human Th9 cells were treated with Calcitriol (100 nM) and Pam₃CSK₄ (2 µg/mL). The number of IL-9+ve cells was determined by flow cytometry. Real-time PCR was used to assess the gene expression. Serum 25(OH)D₃ levels were determined by HPLC. We observed that TLR2 signals via IL-33/ST2 in Th9 cells. Increased TLR2 expression associated with increased IL9 expression and augmented disease severity in RA patients. Calcitriol attenuated TLR2 signaling in murine and human Th9 cells. Low serum vitamin D3 level negatively associated with increased IL-9 and TLR2 expression and disease severity in RA patients. Our data suggest a potential role of calcitriol to ameliorate the disease severity of RA patients.

     

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-β and FoxP3 mRNA. Ιn active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.     

iNKT cells can effectively inhibit IL-6 production by B cells in systemic sclerosis

ObjectiveSystemic sclerosis (SSc) is a connective tissue disease with poorly understood pathogenesis and limited treatment options. Patient mortality is rooted predominantly in the development of pulmonary and cardiac complications. The overactivated immune system is assumed to sustain the inflammatory signature of this autoimmune disease. Here, we investigate the potential of immunoregulatory invariant natural killer T (iNKT) cells to inhibit proinflammatory B cell responses in an in vitro model of inflammation.MethodsB cells from healthy volunteers (n = 17) and patients with SSc (n = 15) were used for functional testing upon lipopolysaccharide (LPS) stimulation in a co-culture system with third-party iNKT cells. Cytokine production was measured with antibody-based immunoassays (ELISA) and intracellular cytokine staining.ResultsiNKT cells strongly inhibited the production of proinflammatory interleukin-6 by B cells upon stimulation with LPS in both healthy volunteers and patients with SSc. In a Transwell assay, cell contact between B cells and iNKT cells proved necessary for this inhibitory effect. Similarly, blocking of CD1d on the surface of B cells abolished the immunoregulatory effect of iNKT cells on B cells. B cell subsets with higher expression of CD1d, namely unswitched memory B cells, were more susceptible to iNKT cell inhibition.ConclusionOur in vitro data underline the potential of iNKT cells in the control of SSc and provide a rationale for the use of novel iNKT cell–based therapeutic strategies in the context of autoimmune diseases.     

Substantial molecular variation and low genetic structure in Kenya’s black rhinoceros: implications for conservation

Kenya’s black rhinoceros population declined by more than 98% from 20,000 individuals in the 1970s to around 400 individuals in 1990 due to the effects of poaching, at which time the surviving individuals were isolated in a series of demographically inviable subpopulations. An initial management exercise translocated the survivors into four high security sanctuaries to control poaching and enhance breeding, and this measure successfully arrested the decline. Subsequently, new sanctuaries were established and the metapopulation size reached 650 animals by 2008. However, translocations and the current management strategy that partitions the metapopulation into ‘montane’ and ‘lowland’ rhinoceros may have substantial consequences at the population level and their impact on population genetic diversity has not been investigated. In this study, 12 of the 16 extant subpopulations were analysed using 408 bp of mitochondrial control region sequence (n = 170) and nine microsatellite loci (n = 145). Both markers detected moderate to high genetic diversity (h = 0.78 ± 0.027, n = 170; H_O = 0.70 ± 0.087, n = 145) consistent with previous studies on Diceros bicornis michaeli. However, mtDNA and nDNA diversity varied substantially between subpopulations. The results suggest that the Masai Mara is more differentiated, inbred and isolated than other subpopulations. It also suggests that there are neither distinct montane and lowland groups nor other detectable historical barriers to gene flow. Instead the large majority of genetic diversity was partitioned at the level of individuals; highlighting the need to conserve as many individuals as possible. Future translocations should consider the genetic profile of individuals and the demographic history of both the donor and recipient subpopulations.     

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4⁺ and CD8⁺ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4⁺ and CD8⁺ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4⁺ and CD8⁺ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4⁺ and CD8⁺ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4⁺ and CD8⁺ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2). Received: 12 November 1999 / Accepted: 2 January 2000     

Ranging behaviour and socio-biology of Eurasian otters (Lutra lutra) on lowland mesotrophic river systems

We examined the spatial structure and socio-biology of a native wild population of Eurasian otters (Lutra lutra) on mesotrophic rivers in a mild temperate climate. Radio-tracking of 20 individuals revealed exclusive intra-sexual adult home-ranges. Adult female home-ranges (7.5 km, SD = 1.5 km, n = 7) were inversely related to river width (, F ₆ = 13.5, P = 0.014) and so appeared to be based on food resources. The aquatic area within adult male home-ranges (30.2 ha, SD = 9.5 ha, n = 5) was greater than that within adult female’s (16.8 ha, SD = 7.0 ha) (t ₁₀ = 2.437, P = 0.035), though this result is inconclusive because some males were tracked on oligotrophic rivers. One adult male expanded its range from 10.2 km to 19.3 km within 5 days of the death of the neighbouring male, suggesting that male home-ranges were heavily influenced by conspecifics.     

Effects of remifentanyl and fentanyl on LPS-induced cytokine release in human whole blood in vitro

Aim The present study sought insight into the effects of remifentanyl and fentanyl on LPS-induced release of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-10 in human whole blood. Methods Whole blood was incubated in the presence and absence of remifentanyl and fentanyl. Effects of remifentanyl and fentanyl on spontaneous and endotoxin (lipopolysaccharide; 100 ng ml⁻¹)-stimulated cytokine release were studied in whole blood from volunteers (n = 10) cultured for 6 h. Results IL-6, TNF-α and IL-10 concentrations in groups added with LPS were significantly higher than those in control group (P < 0.01). IL-6, TNF-α and IL-10 concentrations in activation groups treated with remifentanyl or fentanyl were significantly lower than those in LPS treated group (P < 0.05). There were no significant differences on IL-6,TNF-α and IL-10 concentrations in drug-alone groups compared with control group (P > 0.05). Conclusion Remifentanyl or fentanyl alone has no effects on IL-6, TNF-α and IL-10 production, but could attenuate LPS-induced IL-6,TNF-α and IL-10 production in human whole blood. Remifentanyl and fentanyl could inhibit the expressions of IL-6, TNF-α and IL-10 induced by LPS.