The effect of puberty on diurnal sodium regulation.

The aim of this study was to investigate the impact of sex and puberty stage on circadian changes in sodium excretion, sodium-regulating hormones, and hemodynamics. Thirty-nine healthy volunteers (9 prepuberty boys, 10 prepuberty girls, 10 puberty boys, and 10 puberty girls) were included. They all underwent a 24-h circadian in-patient study under standardized conditions regarding activity, diet, and fluid intake. Blood samples were drawn every 4 h, and the urine was collected in fractions. Blood pressure and heart rate were noninvasively monitored. Atrial natriuretic peptide (ANP), angiotensin II, aldosterone, and renin were measured in blood. Children in puberty had lower plasma levels of renin (P<0.05) and angiotensin II (P<0.05) and a 26% reduction in filtered sodium without changes in sodium excretion compared with prepuberty children. A circadian rhythm in sodium excretion, the renin-angiotensin system, ANP, and blood pressure was found with a midnight ANP peak (P<0.001), a nighttime decrease in hemodynamic parameters (P<0.001), an increase in plasma renin (P<0.001) and angiotensin II (P<0.001), and a decrease in sodium excretion (P<0.001) mainly on the basis of increased sodium reabsorption (P<0.001). The timing of the changes did not depend on sex or puberty group. There is a circadian rhythm of sodium excretion and sodium regulation in 7- to 15-yr-old children. This rhythm is similar in boys and girls. As an important new finding, puberty changes the plasma levels of renin and angiotensin II without changing the amount of sodium excreted or the day to night sodium excretion ratio.

Long-term subarachnoid haemorrhage survivors still die due to cerebrovascular causes.

OBJECTIVE: Subarachnoid haemorrhage (SAH) is associated with sympathetic nervous activation and inflammation. SAH could therefore theoretically be a risk factor for development of cardiovascular disease. The aim of this study was to investigate whether long-term (≥1 year) SAH survivors had an increased risk of death due to cardiovascular causes. MATERIAL & METHODS: SAH patients ≥18 years treated at Umeå University Hospital between 1986 and 2006 were eligible for inclusion. Deceased patients were identified in the Swedish population register. Death certificates from long-term SAH survivors and causes of death in the general population were obtained from the National Board of Health and Welfare, Sweden. The prevalence of comorbidities at the time of SAH was compared with the distribution of cardiovascular risk factors in the northern Sweden MONICA (Multinational Monitoring of Trends and Determinants in Cardiovascular Disease) health survey. Analyses were stratified for age and sex. RESULTS: In the SAH patients, the median year of SAH was 1992 and the median year of death was 2001. The MONICA survey in 1994 and the distribution of deaths in the general population in 2001 were used for comparison. Long-term SAH survivors had, compared to the general population, a significantly increased risk for death due to cerebrovascular disease (P < 0.0001), but not for death due to cardiovascular disease. Hypertension was more common in SAH patients compared to survey participants (P < 0.01). CONCLUSION: Cerebrovascular causes of death were significantly more common in long-term survivors after SAH compared to the general population.

Fat depot-specific mRNA expression of novel loci associated with waist-hip ratio.

OBJECTIVE: We hypothesized that genes within recently identified loci associated with waist-hip ratio (WHR) exhibit fat depot-specific mRNA expression, which correlates with obesity-related traits. METHODS: Adipose tissue (AT) mRNA expression of 6 genes (TBX15/WARS2, STAB1, PIGC, ZNRF3 and GRB14) within these loci showing coincident cis-expression quantitative trait loci was measured in 222 paired samples of human visceral (vis) and subcutaneous (sc) AT. The relationship of mRNA expression levels with obesity-related quantitative traits was assessed by Pearson's correlation analyses. Multivariate linear relationships were assessed by generalized linear regression models. RESULTS: Whereas only PIGC, ZNFR3 and STAB1 mRNA expression in sc AT correlated nominally with WHR (P<0.05, adjusted for age and sex), mRNA expression of all studied genes in at least one of the fat depots correlated significantly with vis and/or sc fat area (P ranging from 0.05 to 4.0 × 10(6), adjusted for age and sex). Consistently, the transcript levels of WARS, PIGC and GRB14 were nominally associated with body mass index (BMI) (P ranging from 0.02 to 9.2 × 10(5), adjusted for age and sex). Moreover, independent of sex, obesity and diabetes status, differential expression between vis and sc AT was observed for all tested genes (P<0.01). Finally, the rs10195252 T-allele was nominally associated with increased GRB14 sc mRNA expression (P=0.025 after adjusting for age, sex and BMI). CONCLUSIONS: Our data including the inter-depot variability of mRNA expression suggests that genes within the WHR-associated loci might be involved in the regulation of fat distribution.

Puberty alters renal water handling.

We investigated the influence of sex and puberty stage on circadian urine production and levels of antidiuretic hormone [arginine vasopressin (AVP)] in healthy children. Thirty-nine volunteers (9 prepuberty boys, 10 prepuberty girls, 10 midpuberty boys, and 10 midpuberty girls) were included. All participants underwent a 24-h circadian inpatient study under standardized conditions regarding Na(+) and fluid intake. Blood samples were drawn every 4 h for measurements of plasma AVP, serum 17-ß-estradiol, and testosterone, and urine was fractionally collected for measurements of electrolytes, aquaporin (AQP)2, and PGE2. We found a marked nighttime decrease in diuresis (from 1.69 ± 0.08 to 0.86 ± 0.06 ml·kg(-1)·h(-1), P < 0.001) caused by a significant nighttime increase in solute-free water reabsorption (TcH2O; day-to-night ratio: 0.64 ± 0.07, P < 0.001) concurrent with a significant decrease in osmotic excretion (day-to-night ratio: 1.23 ± 0.06, P < 0.001). Plasma AVP expressed a circadian rhythm (P < 0.01) with a nighttime increase and peak levels at midnight (0.49 ± 0.05 pg/ml). The circadian plasma AVP rhythm was not influenced by sex (P = 0.56) or puberty stage (P = 0.73). There was significantly higher nighttime TcH2O in prepuberty children. This concurred with increased nighttime urinary AQP2 excretion in prepuberty children. Urinary PGE2 exhibited a circadian rhythm independent of sex or puberty stage. Levels of serum 17ß-estradiol and testosterone were as expected for sex and puberty stage, and no effect on the AVP-AQP2-TcH2O axis was observed. This study found a circadian rhythm of plasma AVP independent of sex and puberty stage, although nighttime TcH2O was higher and AQP2 excretion was more pronounced in prepuberty children, suggesting higher prepuberty renal AVP sensitivity.

Cortisol levels are influenced by sedation in the acute phase after subarachnoid haemorrhage.

BACKGROUND: Subarachnoid haemorrhage (SAH) is a life-threatening condition that may be aggravated by acute pituitary damage and cortisol insufficiency. Robust diagnostic criteria for critical illness-related corticosteroid insufficiency (CIRCI) are lacking. The aim of this study was to assess the frequency of CIRCI in the acute phase (0-240 h) after SAH and to evaluate associations between cortisol levels and clinical parameters (sedation, circulatory failure, gender, age, severity of disease, treatment). CIRCI was defined as a single morning serum cortisol (mSC) < 200 nmol/L. The lower limit for calculated free cortisol (cFC) was set at < 22 nmol/L, and for saliva cortisol at < 7.7 nmol/L. METHODS: Fifty patients were included. Serum/saliva cortisol and corticosteroid-binding globulin were obtained every second morning. A logistic regression model was used for multivariate analysis comparing cortisol levels with clinical parameters. RESULTS: Of the patients, 21/50 (42%) had an mSC < 200 nmol/L and 30/50 (60%) had a cFC < 22 nmol/L. In patients with continuous intravenous sedation, the odds ratio for a mSC to be < 200 nmol/L was 18 times higher (95% confidence interval 4.2-85.0, P < 0.001), and the odds ratio for a cFC to be < 22 nmol/L was 2.4 times higher (95% confidence interval 1.2-4.7, P < 0.05) compared with patients with no continuous intravenous sedation. CONCLUSIONS: Continuous intravenous sedation was significantly associated with cortisol values under defined limits (mSC < 200, cFC < 22 nmol/L). The possibility that sedating drugs per se may influence cortisol levels should be taken into consideration before CIRCI is diagnosed.

The common PPARgamma Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes.

Genetic association studies are viewed as problematic and plagued by irreproducibility. Many associations have been reported for type 2 diabetes, but none have been confirmed in multiple samples and with comprehensive controls. We evaluated 16 published genetic associations to type 2 diabetes and related sub-phenotypes using a family-based design to control for population stratification, and replication samples to increase power. We were able to confirm only one association, that of the common Pro12Ala polymorphism in peroxisome proliferator-activated receptor-gamma(PPARgamma) with type 2 diabetes. By analysing over 3,000 individuals, we found a modest (1.25-fold) but significant (P=0.002) increase in diabetes risk associated with the more common proline allele (85% frequency). Moreover, our results resolve a controversy about common variation in PPARgamma. An initial study found a threefold effect, but four of five subsequent publications failed to confirm the association. All six studies are consistent with the odds ratio we describe. The data implicate inherited variation in PPARgamma in the pathogenesis of type 2 diabetes. Because the risk allele occurs at such high frequency, its modest effect translates into a large population attributable risk-influencing as much as 25% of type 2 diabetes in the general population.

Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in insulin target tissues from three inbred rat strains that differ in diabetes susceptibility. METHODS: Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto-Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (n = 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions. RESULTS: We found 29 significantly differentiated microRNAs (p(adjusted) < 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (p(adjusted) = 0.0005) and miR-27a (p(adjusted) = 0.006) were upregulated in adipose tissue; miR-195 (p(adjusted) = 0.006) and miR-103 (p(adjusted) = 0.04) were upregulated in liver; and miR-10b (p(adjusted) = 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (p = 0.008), miR-27a (p = 0.02) and the previously reported miR-29a (p = 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes. CONCLUSION: The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto-Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.

Interaction between early maternal smoking and variants in TNF and GSTP1 in childhood wheezing.

BACKGROUND: Children exposed to tobacco smoke early in life have a higher risk of wheeze. Individual susceptibility may depend on genetic factors. OBJECTIVE: We studied whether variations in single nucleotide polymorphisms (SNPs) in the TNF, glutathione S transferase P1 (GSTP1) and beta2-adrenoreceptor (ADRB2) genes modify the effect of early maternal smoking on the development of childhood asthma, wheeze and allergic sensitization. METHODS: In the Swedish prospective birth cohort BAMSE (Children, Allergy, Milieu, Stockholm, Epidemiological Survey) (n=4089), data collection included questionnaires to measure tobacco smoke exposure and clinical outcomes up to age 4 and medical examinations with blood sampling for specific IgE measurements and genotyping. We defined early maternal smoking as daily smoking by the mother during pregnancy and/or postnatally. We investigated five TNF, six GSTP1 and three ADRB2 SNPs in 982 selected wheezers and non-wheezers. RESULTS: An interaction with early maternal smoking was found for three TNF SNPs (-857C/T, Intron 1, Intron 3) with respect to early wheeze (up to 2 years of age). For example, the odds ratio (OR) for developing early wheeze related to early maternal smoking was 2.4 [95% confidence interval (CI) 1.6-3.7] in children with a wild-type CC homozygote genotype of the TNF-857 SNP, while no tobacco-related risk was seen in children carrying the rare T allele. A clear dose response was observed in children with the CC genotype, with an OR of 1.3 (95% CI 1.1-1.5) per each additional pack per week smoked by the mother during pregnancy. A suggestive interaction with early maternal smoking was also seen for three GSTP1 SNPs (Intron 5, Intron 6 and Ile105Val) with respect to transient wheeze, but not for ADRB2 and wheeze phenotypes. No effect modifications were observed for allergic sensitization. CONCLUSION: Our results suggest that the risk of early childhood wheeze associated with early maternal smoking may be modified by TNF and GSTP1 polymorphisms.

The enigma of the welfare state: excellent child health prerequisites--poor subjective health.

UNLABELLED: The rate of subjective health complaints among Swedish children is increasing by age and over time, and more so than among children in other Scandinavian countries. In contrast, the somatic health and prerequisites for wellbeing are excellent. This paradoxical situation, The Enigma of the Welfare State, is the focus of this viewpoint. We argue that one important background factor may be late adverse effects of the welfare society itself and some of its inherent values. We have identified several possible pathways. We have given them names of diseases--on the society level--like health obsession, stress panic, welfare apathy and hyper-individualism. Together with other factors such as a dysfunctional school and an unsatisfactory labour market for youth, these diseases are involved in an interplay that is constantly inducing anxiety and low self-esteem. CONCLUSION: The gradually deteriorating self-reported health among Swedish youth may, to some degree, be explained as a late adverse effect of the welfare society itself and its inherent values.

Genome-wide methylation profiling in granulosa lutein cells of women with polycystic ovary syndrome (PCOS).

Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder amongst women of reproductive age, whose aetiology remains unclear. To improve our understanding of the molecular mechanisms underlying the disease, we conducted a genome-wide DNA methylation profiling in granulosa lutein cells collected from 16 women suffering from PCOS, in comparison to 16 healthy controls. Samples were collected by follicular aspiration during routine egg collection for IVF treatment. Study groups were matched for age and BMI, did not suffer from other disease and were not taking confounding medication. Comparing women with polycystic versus normal ovarian morphology, after correcting for multiple comparisons, we identified 106 differentially methylated CpG sites with p-values <5.8 × 10-8 that were associated with 88 genes, several of which are known to relate either to PCOS or to ovarian function. Replication and validation of the experiment was done using pyrosequencing to analyse six of the identified differentially methylated sites. Pathway analysis indicated potential disruption in canonical pathways and gene networks that are, amongst other, associated with cancer, cardiogenesis, Hedgehog signalling and immune response. In conclusion, these novel findings indicate that women with PCOS display epigenetic changes in ovarian granulosa cells that may be associated with the heterogeneity of the disorder.

Population structure in contemporary Sweden--a Y-chromosomal and mitochondrial DNA analysis.

A population sample representing the current Swedish population was analysed for maternally and paternally inherited markers with the aim of characterizing genetic variation and population structure. The sample set of 820 females and 883 males were extracted and amplified from Guthrie cards of all the children born in Sweden during one week in 2003. 14 Y-chromosomal and 34 mitochondrial DNA SNPs were genotyped. The haplogroup frequencies of the counties closest to Finland, Norway, Denmark and the Saami region in the north exhibited similarities to the neighbouring populations, resulting from the formation of the Swedish nation during the past millennium. Moreover, the recent immigration waves of the 20th century are visible in haplogroup frequencies, and have led to increased diversity and divergence of the major cities. Signs of genetic drift can be detected in several counties in northern as well as in southern Sweden. With the exception of the most drifted subpopulations, the population structure in Sweden appears mostly clinal. In conclusion, our study yielded valuable information of the structure of the Swedish population, and demonstrated the usefulness of biobanks as a source of population genetic research. Our sampling strategy, nonselective on the current population rather than stratified according to ancestry, is informative for capturing the contemporary variation in the increasingly panmictic populations of the world.

Mechanisms of inactivation of MLH1 in hereditary nonpolyposis colorectal carcinoma: a novel approach.

Mutations in the DNA mismatch repair gene MLH1 are a major cause of hereditary nonpolyposis colorectal cancer (HNPCC). No mutant phenotype is observed before the wild-type (wt) allele is somatically inactivated in target tissue. We addressed the mechanisms of MLH1 inactivation in 25 colorectal (CRC) and 32 endometrial cancers (ECs) from MLH1 mutation carriers (Mut1, in-frame genomic deletion; Mut2, out-of-frame splice site mutation; Mut3, missense mutation). By a quantitative method, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF), utilizing four intragenic single nucleotide polymorphisms and mutations, loss of heterozygosity (LOH) was present in 31/57 (54.4%) of tumors. The wt allele displayed LOH more often than the mutant allele (23/57 vs 8/57, P=0.006). For Mut1, LOH was more frequent in CRC than EC (10/11 vs 1/13, P<0.0001), whereas Mut2 and Mut3 displayed opposite LOH pattern. Moreover, although wt LOH predominated in CRC irrespective of the predisposing mutation, LOH often affected the mutant allele in EC from Mut2 and Mut3 carriers (6/19, 31.6%). MLH1 promoter methylation, which reflected a more widespread hypermethylation tendency, occurred in 4/55 (7.3%) of tumors and was inversely associated with LOH. In conclusion, the patterns of somatic events (LOH and promoter methylation) differ depending on the tissue and germline mutation, which may in part explain the differential tumor susceptibility of different organs in HNPCC. MALDI-TOF provides a novel approach for the detection and quantification of LOH.

Elevated serum levels of estradiol, dihydrotestosterone, and inhibin B in adult males born small for gestational age.

CONTEXT: Prenatal growth restriction may affect future fertility in both females and males. Studies have shown that growth-retarded male rats have different sexual behavior and disturbed steroidogenesis. OBJECTIVE: We hypothesized that adult human males born small for gestational age (SGA) have an altered sex hormone profile. DESIGN, SETTING, AND PATIENTS: Twenty-five adult males born SGA with median birth weight -2.2 sd scores (SDS) and birth length -2.4 SDS were studied. Median age was 23.1 yr and final height -0.5 SDS. They were compared with 44 male controls with median age 20.5 yr and final height 0.4 SDS. MAIN OUTCOME MEASURE: The primary outcome before the study started was 17beta-estradiol (E(2)) levels in SGA males. RESULTS: The SGA group showed significantly higher median levels of E(2), 17.9 pg/ml (P < 0.001), and dihydrotestosterone (DHT), 0.543 ng/ml (P < 0.05), compared with controls, 12.6 pg/ml and 0.423 ng/ml, respectively. Testosterone (T) levels did not differ between groups. E(2) to T ratio correlated negatively to birth weight (r = -0.40, P < 0.01) and birth length (r = -0.44, P < 0.001). DHT to T ratio correlated negatively to birth weight (r = -0.51, P < 0.001) and birth length (r = -0.38, P < 0.01). Males born SGA also had significantly higher median levels of inhibin B, 164 pg/ml (P < 0.05), compared with controls, 137 pg/ml. Inhibin B correlated negatively to birth length (r = -0.34, P < 0.01). CONCLUSION: SGA males of normal stature have higher levels of E(2), DHT, and inhibin B than controls, indicating a disturbed steroid synthesis or metabolism. Aromatase activity, calculated as E(2) to T ratio, and 5alpha-reductase activity, calculated as DHT to T ratio, is negatively correlated to size at birth.

Androgens in women after allogeneic hematopoietic cell transplantation: impact of chronic GvHD and glucocorticoid therapy.

Low androgen levels may contribute to sexual dysfunction in women after allogeneic hematopoietic cell transplantation (alloHCT). However, data on serum androgens in women after alloHCT are limited. The aim of this study was to assess androgen levels and their association with chronic GvHD (cGvHD) and glucocorticoid (GC) therapy. Included were 65 allografted women, 33 with cGvHD, and 23 of these were on GC therapy. Controls were 94 healthy, age-matched women. Supportive study groups were women after autologous HCT (autoHCT; n=20) and non-transplanted women on GC therapy (n=26). Compared with controls, free testosterone (free T) and dehydroepiandrosterone sulfate (DHEAS) levels were lower in both the alloHCT group and GC groups; P<0.0001 and P<0.05, respectively. Androgens in the autoHCT group were similar or higher than controls. In the subgroup of alloHCT patients without cGvHD, free T was similar to controls (7.2 vs 8.6 pmol/L; P=0.42), whereas DHEAS levels was lower than controls (1.7 vs 2.5 µmol/L; P=0.008). Compared with controls, cGvHD without GC (n=10) was associated with lower free T and DHEAS; P=0.004 and P=0.0004, respectively). The lowest androgen levels were seen in women with both cGvHD and GC therapy. In conclusion, low serum androgens were associated with cGvHD and GC therapy, prompting for studies assessing a possible association between low androgens and sexual dysfunction and quality of life in allografted women.

Phenylketonuria screening registry as a resource for population genetic studies.

BACKGROUND: Neonatal screening for metabolic diseases, involving samples stored on filter paper (Guthrie spots), provides a potential resource for genetic epidemiological studies. OBJECTIVE: To develop a method to make these dried blood spots available for large scale genetic epidemiology. METHODS: DNA from untraceable Guthrie spots was extracted using a saponin and chelex-100 based method and preamplified by improved primer preamplification. Analyses were done on 38 samples each of fresh, 10, and 25 year old Guthrie spots and the success rate determined for PCR amplification for five amplicon lengths. RESULTS: The method was applicable even on 25 year old samples. The success rate was 100% for 100 bp amplicons and 80% for 396 bp amplicons. Ninety four Guthrie samples were genotyped, including carriers of two different PKU mutations; all carriers were found (six R158Q, four R252W), with no false positives. Finally, 2132 anonymous samples from the Swedish PKU registry were extracted and preamplified and the allele frequencies of APOepsilon4, PPARgamma Pro12Ala, and the CCR5 32 bp deletion determined. Local variations in allele frequencies suggested subpopulation structuring. There was a significant difference (p<0.01) in regional allele frequencies for the CCR5 32 bp deletion in the Swedish population. CONCLUSION: Whole genome amplification makes it feasible to conduct large genetic epidemiological studies using PKU screening registries.

Global analysis of uniparental disomy using high density genotyping arrays.

BACKGROUND: Uniparental disomy (UPD), the inheritance of both copies of a chromosome from a single parent, has been identified as the cause for congenital disorders such as Silver-Russell, Prader-Willi, and Angelman syndromes. Detection of UPD has largely been performed through labour intensive screening of DNA from patients and their parents, using microsatellite markers. METHODS: We applied high density single nucleotide polymorphism (SNP) microarrays to diagnose whole chromosome and segmental UPD and to study the occurrence of continuous or interspersed heterodisomic and isodisomic regions in six patients with Silver-Russell syndrome patients who had maternal UPD for chromosome 7 (matUPD7). RESULTS: We have devised a new high precision and high-throughput computational method to confirm UPD and to localise segments where transitions of UPD status occur. Our method reliably confirmed and mapped the matUPD7 regions in all patients in our study. CONCLUSION: Our results suggest that high density SNP arrays can be reliably used for rapid and efficient diagnosis of both segmental and whole chromosome UPD across the entire genome.

Lymphokine-activated killer function following autologous bone marrow transplantation for refractory hematological malignancies.

Disease recurrence remains the major factor which limits the success of autologous bone marrow transplantation (ABMT) for refractory hematological malignancies. The administration of interleukin 2 (IL2) with or without ex vivo generated lymphokine-activated killer (LAK) cells represents a potential approach to eradicating residual disease after ABMT. However, since LAK precursor activity is radiosensitive, high dose chemoradiotherapy may abrogate LAK function and preclude clinical responsiveness to IL2 after ABMT. Furthermore, since lymphocyte subsets which mediate LAK activity may recover at different rates after ABMT, LAK cells may be phenotypically and/or functionally altered after ABMT. To determine whether IL2 responsive LAK precursor cells are present in the circulation after ABMT, peripheral blood mononuclear cells (PBMC) from 21 patients with acute leukemia or lymphoma were tested for IL2-inducible LAK activity 17-83 days after ABMT. Cells were cultured with IL2 (1000-2000 units/ml) for 4 or 5 days and then tested for cytolytic activity and/or cell phenotype. LAK activity against the Daudi cell line was detected in every PBMC sample from every patient at every time point tested. The Raji cell line and a fresh allogeneic ovarian carcinoma were also lysed by LAK cells generated after ABMT. In the subgroup of patients transplanted for non-Hodgkin's lymphoma, LAK precursor activity appeared comparable to that of healthy controls. Culture with IL2 resulted in increased mean IL2 receptor expression in lymphocytes from patients after ABMT (3.1-9.9%) and from healthy controls (3.1-12.0%). After culture with IL2, the percentage of cells bearing the natural killer cell-associated Leu-19 determinant was significantly higher in patient PBMC than in normal control PBMC (28.3 versus 8.7%). Positive and negative cell selection by fluorescence sorting after culture with IL2 revealed that most of the LAK activity after ABMT was mediated by the Leu-19+ cells. Although CD5+ T-cells were devoid of LAK activity, a subset LAK effectors was CD8+. Thus, LAK activity is rapidly reconstituted after ABMT and is mediated by cells phenotypically similar to those in normal controls. These results support the feasibility of IL2 +/- LAK as consolidative immunotherapy after ABMT.

Recombinant interleukin 2 toxicity, pharmacokinetics, and immunomodulatory effects in a phase I trial.

Twenty-two patients with refractory malignancies were treated with four escalating weekly doses of recombinant interleukin 2 (IL2), given either i.v. by 2- or 24-h infusion, or s.c. A 1-wk washout period between each dose of IL2 was provided for the evaluation for pharmacokinetic and immunomodulatory effects. The maximum i.v. dose was 30 X 10(6) units; the dose-limiting toxicities were fever, flu-like symptoms, and hypotension. The maximum s.c. dose was 3 X 10(6) because of volume limitations with s.c. injection. No tumor regression was seen. During infusions of 3 X 10(6) units over 2 h or 24 h, serum IL2 levels greater than or equal to 223 units/ml or 16 units/ml were maintained, respectively; with s.c. injection of 3 X 10(6) units, levels greater than 20 units/ml were maintained for 9 h. Marked lymphopenia was observed 24 h after the initiation of IL2 doses which was completely reversible when measured prior to the next dose. The lymphopenia was nonselective; T- and B-lymphocytes decreased in an IL2 dose-dependent manner, without consistent change in the OKT4:OKT8 ratio. No change was detected in monocyte expression of HLA-Dr or T-cell expression of the IL2 receptor. The in vitro generation of lymphokine-activated killer cytotoxicity decreased sharply and transiently shortly after i.v. doses. Mitogen responsiveness, delayed-type hypersensitivity, natural killer cytotoxicity, and mixed-lymphocyte reactivity were unchanged or decreased transiently shortly after IL2 doses. These studies help define the bioavailability of IL2 by i.v. or s.c. routes, and they will aid in the design of studies utilizing daily doses of IL2.

Enhancement by interleukin 4 of interleukin 2- or antibody-induced proliferation of lymphocytes from interleukin 2-treated cancer patients.

Systemic interleukin 2 (IL-2) and IL-2-activated lymphocytes have induced tumor regression in some cancer patients. The IL-2-activated cells have usually been generated by obtaining peripheral blood mononuclear cells (PBMC) from cancer patients shortly after systemic IL-2 therapy and culturing them with IL-2 in vitro. In an effort to augment the ex vivo generation of such cells preactivated in vivo, we examined the proliferative responses of PBMC from IL-2-treated cancer patients to several proliferative signals including IL-2, interleukin 4 (IL-4), and mitogenic antibodies to CD3 and CD28. Although much is known about the response of normal PBMC to these signals, the possibility was considered that the response of lymphocytes preactivated by IL-2 in vivo might differ from that of normal PBMC. Accordingly, PBMC obtained from ten normal, healthy controls and from 17 patients with advanced cancer 1 to 3 days after systemic IL-2 therapy were cultured for 4 days with IL-4 (1000 units/ml) and/or IL-2 (10 units/ml or 1000 units/ml) or with combinations of IL-4 and anti-CD3 +/- anti-CD28, and they were then tested for proliferation by [3H]thymidine incorporation. IL-4 failed to induce proliferation of normal PBMC and inhibited IL-2-induced proliferation, whereas IL-4 alone induced proliferation in PBMC from five of 11 IL-2-treated patients and did not inhibit but augmented the proliferation induced by IL-2 (10 units/ml and 1000 units/ml) in PBMC from six of nine patients and five of 11 patients, respectively. Anti-CD3 induced proliferation in PBMC from eight of nine patients, and the proliferation was consistently augmented by coculture with anti-CD28. Finally, IL-4 significantly augmented the proliferative responses of PBMC from IL-2-treated patients to anti-CD3, as well as to the combination of anti-CD3 and anti-CD28. Thus, in PBMC from IL-2-treated cancer patients, IL-4 enhanced the in vitro proliferation induced by IL-2 or by anti-CD3 +/- anti-CD28. The results suggest that IL-4 and/or mitogenic antibodies may be useful in augmenting the ex vivo generation of lymphocytes for clinical adoptive immunotherapy.

Induction of lymphokine-activated killer activity by interleukin 4 in human lymphocytes preactivated by interleukin 2 in vivo or in vitro.

In an attempt to augment the generation of human cytotoxic effector cells for potential cancer therapy with interleukin 2 (IL2) and lymphokine-activated killer (LAK) cells, the effect of interleukin 4 (IL4) on LAK cell induction was studied. In normal human peripheral blood lymphocytes (PBL), IL4 does not induce LAK activity and inhibits LAK induction by IL2. However, since lymphocyte activation, such as with antigen or mitogen, can render them responsive to IL4, the ability of IL4 to induce LAK activity in lymphocytes preactivated in vivo or in vitro with IL2 was investigated. PBL obtained from 12 patients with advanced cancer 1 to 3 days after IL2 therapy and from eight healthy control subjects were cultured 4 to 5 days with or without IL4 and/or IL2 and then tested for LAK activity as assessed by lysis of Daudi in a 4-h 51Cr release assay. In normal PBL, IL4 failed to induce LAK activity and consistently inhibited LAK induction by a suboptimal concentration of IL2 (10 units/ml). By contrast, IL4 induced LAK activity in PBL from seven of twelve IL2-treated patients and augmented LAK induction by the suboptimal IL2 in PBL from five of twelve IL2-treated patients. With an optimal LAK-inducing concentration of IL2 (1000 units/ml), IL4 less consistently inhibited LAK induction in normal PBL and had a variable effect upon LAK induction in PBL from IL2-treated patients. IL4 induced LAK activity in PBL obtained from a cancer patient after, but not before, systemic IL2 therapy. Similarly, IL4 induced LAK activity in normal PBL only after they had been preincubated with IL2. Thus, IL4 induces LAK activity in lymphocytes preactivated by IL2 in vivo or in vitro. Fluorescence-activated cell sorting revealed that the LAK activity, whether induced by IL4 or by IL2, was mediated largely by non-T (CD5-) natural killer-like (CD56+) cells. The results suggest a regulatory relationship between IL2 and IL4 in the induction and/or maintenance of LAK activity, which might be exploited to augment the generation of cytotoxic cells for lymphokine-mediated immunotherapy of human cancer.