A European consensus for the evaluation and management of infants with an equivocal diagnosis following newborn screening for cystic fibrosis.

Screening newborns for cystic fibrosis (CF) is considered to be an ethical undertaking in regions with a significant incidence of the condition. Current screening protocols result in recognition of infants with an equivocal diagnosis. A survey of European practice suggested inconsistencies in the evaluation and management of these infants. We have undertaken a consensus process using a modified Delphi method. This has enabled input of CF specialists from a wide geographical area to a rigorous process that has provided a clear pathway to a consensus statement. A core group produced 21 statements, which were modified over a series of three rounds (including a meeting arranged at the European CF Conference). A final document of 19 statements was produced, all of which achieved a satisfactory level of consensus. The statements cover four themes; sweat testing, further assessments and investigations, review arrangements and database. This consensus document will provide guidance to CF specialists with established screening programmes and those who are in the process of implementing newborn screening in their region.

Effects of experimentally induced panic attacks on neuroimmunological markers.

Since little is known concerning regulation of immunological parameters in rapid changing psychiatric states like panic attacks, we measured cytokines at different time points in healthy subjects, which underwent experimental panic induction using the CCK-4 paradigm. Apart from a challenge related IL-6 increase, we could not observe any changes of neuroimmunological markers in relation to acute anxiety with regard to time and group. Herein we conducted for the first time a new approach to immunological research in panic disorder, suggesting immune changes are more related to long term disease stress.

Some cases of common variable immunodeficiency may be due to a mutation in the SBDS gene of Shwachman-Diamond syndrome.

Known genetic defects currently account for only a small proportion of patients meeting criteria for 'probable' or 'possible' common variable immunodeficiency (CVID). A 59-year-old male with a 12-year history of CVID on intravenous immunoglobulin (IVIG) is presented who developed bronchiectasis, cytopenias and malabsorption that are recognized complications of CVID. Work-up for his malabsorption suggested the possibility of Shwachman-Diamond syndrome, confirmed by mutation testing. With the identification of the molecular defect in Shwachman-Diamond syndrome (SDS), it is becoming clear that not all SDS patients have the prominent features of neutropenia or pancreatic malabsorption. A meta-analysis of published immunological defects in SDS suggests that four of 14 hypogammaglobulinaemic SDS patients meet criteria for 'possible' CVID. Mutations in the SBDS gene may therefore be the fifth identified molecular defect in CVID.

The immune-mediated alteration of serotonin and glutamate: towards an integrated view of depression.

Beside the well-known deficiency in serotonergic neurotransmission as pathophysiological correlate of major depression (MD), recent evidence points to a pivotal role of increased glutamate receptor activation as well. However, cause and interaction of these neurotransmitter alterations are not understood. In this review, we present a hypothesis integrating current concepts of neurotransmission and hypothalamus-pituitary-adrenal (HPA) axis dysregulation with findings on immunological alterations and alterations in brain morphology in MD. An immune activation including increased production of proinflammatory cytokines has repeatedly been described in MD. Proinflammatory cytokines such as interleukin-2, interferon-gamma, or tumor necrosis factor-alpha activate the tryptophan- and serotonin-degrading enzyme indoleamine 2,3-dioxygenase (IDO). Depressive states during inflammatory somatic disorders are also associated with increased proinflammatory cytokines and increased consumption of tryptophan via activation of IDO. An enhanced consumption of serotonin and its precursor tryptophan through IDO activation could well explain the reduced availability of serotonergic neurotransmission in MD. An increased activation of IDO and its subsequent enzyme kynurenine monooxygenase by proinflammatory cytokines, moreover, leads to an enhanced production of quinolinic acid, a strong agonist of the glutamatergic N-methyl-D-aspartate receptor. In inflammatory states of the central nervous system, IDO is mainly activated in microglial cells, which preferentially metabolize tryptophan to the NMDA receptor agonist quinolinic acid, whereas astrocytes - counteracting this metabolism due to the lack of an enzyme of this metabolism - have been observed to be reduced in MD. Therefore the type 1/type 2 immune response imbalance, associated with an astrocyte/microglia imbalance, leads to serotonergic deficiency and glutamatergic overproduction. Astrocytes are further strongly involved in re-uptake and metabolic conversion of glutamate. The reduced number of astrocytes could contribute to both, a diminished counterregulation of IDO activity in microglia and an altered glutamatergic neurotransmission. Further search for antidepressant agents should take into account anti-inflammatory drugs, for example, cyclooxygenase-2 inhibitors, might exert antidepressant effects by acting on serotonergic deficiency, glutamatergic hyperfunction and antagonizing neurotoxic effects of quinolinic acid.

The cyclooxygenase-2 inhibitor celecoxib has therapeutic effects in major depression: results of a double-blind, randomized, placebo controlled, add-on pilot study to reboxetine.

Signs of an inflammatory process, in particular increased pro-inflammatory cytokines and increased levels of prostaglandine E(2) (PGE(2)), have repeatedly been described in major depression (MD). As cyclooxygenase-2 (COX-2) inhibitors inhibit the PGE(2) production and the production of pro-inflammatory cytokines, we performed a therapeutic trial with the COX-2 inhibitor celecoxib. In a prospective, double-blind, add-on study, 40 patients suffering from an acute depressive episode were randomly assigned to either reboxetine and celecoxib or to reboxetine plus placebo. After a wash-out period, 20 patients received 4-10 mg reboxetine plus placebo and 20 received reboxetine plus 400 mg celecoxib for 6 weeks. The treatment effect was calculated by analysis of variance. There were no significant differences between groups in age, sex, duration or severity of disease or psychopathology, or reboxetine dose or plasma levels. Over 6 weeks, both groups of patients showed significant improvement in scores of the Hamilton Depression Scale. However, the celecoxib group showed significantly greater improvement compared to the reboxetine-alone group. Additional treatment with celecoxib has significant positive effects on the therapeutic action of reboxetine with regard to depressive symptomatology. Moreover, the fact that treatment with an anti-inflammatory drug showed beneficial effects on MD indicates that inflammation is related to the pathomechanism of the disorder, although the exact mechanisms remain to become elucidated.

Evidence for an altered tryptophan metabolism in fibromyalgia.

Fibromyalgia (FM) is a prevalent syndrome with chronic pain and a hypothesized underlying disturbance of the tryptophan (TRP) metabolism. We performed a tryptophan depletion (TD) test in 17 FM patients and 17 controls. TRP, 5-hydroxyindoleacetic acid (5-HIAA), kynurenine (KYN), and interleukin-6 (IL-6) were measured. Additionally pain perception was monitored in the FM patients. FM patients and controls exhibited a decrease of TRP and KYN during TD. 5-HIAA levels also decreased in all controls and in 11 FM patients, but showed a marked increase in 6 FM patients. IL-6 significantly increased during TD in the patients, but not in the controls. Pain perception was not affected in the FM patients. These data demonstrate an altered TRP metabolism in a subgroup of FM patients, where the TD seems to activate 5-HT metabolism. Our findings may have diagnostic as well as therapeutic implications in the field of fibromyalgia.

TGF-beta(1) genotype and accelerated decline in lung function of patients with cystic fibrosis.

BACKGROUND: Polymorphisms in transforming growth factor (TGF)-beta(1) associated with variations in cytokine levels are linked to fibrosis in a number of tissues. However, the contribution of this cytokine to organ fibrosis in patients with cystic fibrosis is presently unclear. This study was undertaken to examine the association between TGF-beta(1) gene polymorphisms and the development of pulmonary dysfunction in patients with cystic fibrosis. METHODS: Polymorphisms in the TGF-beta(1) gene defining amino acids of codons 10 and 25 were determined by ARMS-PCR using DNA stored on 171 Caucasian patients who were homozygous for the DeltaF508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Clinical information on the patients was obtained from medical records. RESULTS: Patients with cystic fibrosis of a TGF-beta(1) high producer genotype for codon 10 had more rapid deterioration in lung function than those with a TGF-beta(1) low producer genotype. The relative risk of accelerated decline in forced expiratory volume in one second (FEV(1)) to 50% predicted and forced vital capacity (FVC) to 70% predicted of patients with a high producer genotype was 1.74 (95% CI 1.11 to 2. 73) compared with 1.95 (95% CI 1.24 to 3.06) for those with a low producer genotype. DISCUSSION: TGF-beta(1) genotypes may have a role in mediating pulmonary dysfunction in patients with cystic fibrosis. Further work is required to determine whether inhibition of TGF-beta(1) activity in these patients may slow disease progression.

Cellular and humoral immune system in schizophrenia: a conceptual re-evaluation.

Immune alterations in schizophrenia have been described for decades. However, modern immunological methods and new insights into the highly developed and functionally differentiated immune system allow an integrative view of both the older and also more recent findings of immunological abnormalities in schizophrenia. The conceptual advances in immunology require the re-evaluation of elder immunological findings in schizophrenia. In this overview, recent advances in immunological research regarding the differentiation between T-Helper-1 and T-Helper-2 cells and between the so-called specific and unspecific arms of the immune system are discussed. The unspecific "innate" immune system shows signs of an over-activation in unmedicated schizophrenic patients, as increased monocytes and gamma delta-cells point to. Increased levels of Interleukin-6 (IL-6) and the activation of the IL-6 system in schizophrenia might be the result of the activation of monocytes/macrophages, too. In contrast, several parameters of the specific cellular immune system are blunted, e.g. the decreased T-helper-1 (TH-1) related immune parameters in schizophrenic patients, both in vitro and in vivo. It seems that a TH-1-TH-2 imbalance with a shift to the TH-2 system is associated with schizophrenia. During therapy with antipsychotics, the specific TH-1 related immune answer becomes activated, but the B-cell system and the antibody production become activated too.

CSF and serum levels of soluble interleukin-6 receptors (sIL-6R and sgp130), but not of interleukin-6 are altered in multiple sclerosis.

Interleukin-6 (IL-6) has recently been implicated in multiple sclerosis (MS), since IL-6 deficient mice were resistant to a demyelinating form of experimental autoimmune encephalomyelitis and IL-6 expression was upregulated in MS. The cytokine IL-6 and its action mediating soluble receptors (sIL-6R and sgp130) were measured in cerebrospinal fluid (CSF) and serum of 61 MS patients and 39 controls. In the presence of unchanged IL-6 concentrations, sIL-6R and sgp130 serum levels were significantly increased in MS and correlated with disease severity. Furthermore, sgp130 CSF levels were decreased in MS, suggesting a possibly altered IL-6 regulation in the CSF.

Discriminant power of combined cerebrospinal fluid tau protein and of the soluble interleukin-6 receptor complex in the diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) still can only be definitively diagnosed with certainty by examination of brain tissue. There is a great need for a noninvasive, sensitive and specific in vivo test for AD. We combined cerebrospinal fluid analyses of tau protein (levels were significantly increased in AD patients [p=0.0001]), a putative marker of neuronal degeneration, with components of the soluble interleukin-6 receptor complex (sIL-6RC: IL-6, soluble IL-6 receptor and soluble gp130), putative markers of neuroregulatory and inflammatory processes in the brain. A stepwise multivariate discriminant analysis revealed that tau protein and soluble gp130 (levels were significantly reduced in AD subjects [p=0.007]), the affinity converting and signal-transducing receptor of neuropoietic cytokines, maximized separation between the investigated groups. The discriminant function predicted 23 of 25 clinically diagnosed AD patients (sensitivity 92%) with mild to moderate dementia correctly as having AD. Furthermore, 17 of 19 physically and cognitively healthy age-matched control subjects (specificity 90%) were accurately distinguished by this test. Later predicting with the jackknife procedure each case in turn through the remaining patient group, the discriminant function remained stable. Our data suggest that multivariate discriminant analysis of combined CSF tau protein and sIL-6RC components may add more certainty to the diagnosis of AD, however, the method will need to be extended to an independent group of patients, comparisons and control subjects to assess the true applicability.

Interleukin-6 and the soluble IL-6 receptor are decreased in cerebrospinal fluid of geriatric patients with major depression: no alteration of soluble gp130.

Interleukin-6 (IL-6) is hypothesized to play an important role in the interaction between immune mechanisms and the central nervous system. We investigated whether cerebrospinal fluid (CSF) concentrations of interleukin-6 (IL-6), the soluble IL-6 receptor (sIL-6R) and the soluble form of the signal transducing and affinity converting receptor gp130 (sgp130) are altered in geriatric patients with major depression (MD). In 20 geriatric patients with MD and 20 age-matched healthy control subjects CSF concentrations of the three components of the sIL-6R complex were analyzed by enzyme-linked immunosorbent assays (ELISA). All patients except one were treated with psychotropic drugs. We found statistically significant decreased CSF concentrations of IL-6 (P<0.001) and of the sIL-6R (P<0.001) of patients with MD. Levels of sgp130 showed no statistically significant difference between patients and controls.

Relationship of substance P, 5-hydroxyindole acetic acid and tryptophan in serum of fibromyalgia patients.

The serotonergic system has repeatedly been discussed to be involved in the pathophysiology of fibromyalgia (FM), which is a syndrome of widespread pain and sleep disturbance. Elevated levels of substance P (SP), a mediator of nociception, have been described in FM. In this study the possible relationship between SP and serotonin (5-HT) together with its precursor tryptophan (TRP) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) was evaluated in 51 serum samples of fibromyalgia patients. These parameters were compared with clinical data such as pain intensity or sleep quality. A strong negative correlation between SP and 5-HIAA (P = .000) as well as between SP and TRP (P = .009) could be demonstrated. High serum concentrations of 5-HIAA and TRP showed a significant relation to low pain scores (5-HIAA: P = .030; TRP: P = .014). Moreover, 5-HIAA was strongly related to good quality of sleep (P = .000), while SP was related to sleep disturbance (P = .005). These data are valid to support the hypothesis of a systemic involvement of 5-HT and SP in fibromyalgia.

Melanoma-associated adhesion molecule MUC18/MCAM (CD146) and transcriptional regulator mader in normal human CNS.

The proteins MUC18 and Mader have been identified as markers of tumor progression in melanoma cells. MUC18, also known as MCAM (melanoma cell adhesion molecule) and as CD146 (endothelial antigen), is a cell adhesion molecule belonging to the immunoglobulin superfamily. Mader is a transcriptional regulator shown to negatively regulate EGR-1. As it is known that neoplastic cells of neuroectodermal origin frequently express neuron-specific molecules, we studied whether these melanoma-associated antigens are found in normal CNS tissue. We investigated the expression of MUC18/MCAM and Mader in adult human post mortem CNS tissue by immunohistochemistry, immunoblot and two-dimensional gel electrophoresis. Our results show that Mader is preferentially expressed on neurons and glial cells and that the adhesion protein MUC18/MCAM is mainly expressed on vasculature within the CNS. These observations may have important implications for further studies investigating their possible roles in cell adhesion and proliferation control within the CNS.

Detection of five novel mutations of the cystic fibrosis transmembrane regulator (CFTR) gene in Pakistani patients with cystic fibrosis: Y569D, Q98X, 296+12(T>C), 1161delC and 621+2(T>C).

We analysed DNA samples from 26 Pakistani patients with cystic fibrosis (CF) living in the United Kingdom (14 from patients residing in the north west of England, who were referred directly to the North West Regional Molecular Genetics Laboratory, and 12 from other regional molecular genetics laboratories). Of 56 mutations seen in native U.K. CF patients, only DeltaF508, R709X, and 2184insA were detected in the Pakistani patients. Combined SSCP/Heteroduplex analysis, DGGE, and direct DNA cycle sequencing revealed five novel mutations: Y569D, Q98X, 296+12(T>C), 1161delC, and 621+2(T>C), which appear to be specific to Pakistani CF families. In addition, a novel polymorphism, 297-67(A/C), and three previously described rare mutations, 1525-1(G>A), R560S, and 1898+1(G>T), were detected. In the 14 Pakistani CF patients from the north west of England, DeltaF508 accounted for approximately 32% (9/28 chromosomes) and the overall detection rate of CF mutations in this group was approximately 86% (24/28 chromosomes).

Adapting in situ polymerase chain reaction for genotyping of cells in suspension.

An approach is described for in situ polymerase chain reaction (ISPCR) based on cycling primed in situ synthesis (PRINS) conditions defined for alpha-satellite DNA. Using blood cell preparations subjected to limited fixation with paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of paired nuclear spots corresponding to specific centromeres. Using longer cycling regimens, primers for single copy genes also generated paired nuclear spots in a primer-pair--specific manner. In this context, the amplification refractory mutation system (ARMS) was evaluated for in situ applications. In ARMS, allele-specific primers are used in such a manner that PCR proceeds only when an exact 3' match between annealed primer and template is recognized by DNA polymerase. Using normal and mutant primers for the delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extraction and reamplification with ARMS primers, it was shown that amplicon accumulates in cells in the expected primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that nonspecific signal is due to primer dimer formation, especially in the absence of true template DNA. The impact of primer dimer formation in generating a false-positive signal is discussed. The method described here enables a cell population to be analyzed for a given point mutation.

Interleukin-6 is not altered in cerebrospinal fluid of first-degree relatives and patients with Alzheimer's disease.

We investigated interleukin-6 (IL-6) levels in cerebrospinal fluid (CSF) of 25 patients with clinically diagnosed sporadic Alzheimer's disease (AD) and 19 healthy control subjects (HC). For comparison 19 clinically healthy subjects with at least one first-degree relative with clinical or autopsy confirmed AD (CF/AD) were examined. CSF levels of IL-6 did not show statistically significant differences between AD patients, CF/AD and HC subjects. There was no correlation between age, gender, age of onset, degree of cognitive impairment, blood-brain barrier dysfunction and IL-6 values. We could not demonstrate altered CSF concentrations of IL-6 that may indicate an inflammatory response or capability to support neuronal survival in the central nervous system (CNS) of first-degree relatives and patients with AD. We suggest that combined measurement of all parameters of the IL-6-receptor complex could yield more insight in a probably altered IL-6 function.

Cystic fibrosis mutations in Romania.

UNLABELLED: We have investigated the genotype in 32 children with cystic fibrosis from Romania. The diagnosis of cystic fibrosis was made on the basis of typical clinical findings and sweat electrolyte levels using the pilocarpine iontophoresis method. CONCLUSION: Genetic analysis of 32 children with cystic fibrosis from Romania showed a 25% incidence of DeltaF508 mutation and a 64.5% incidence of unknown mutations, 5 other known mutations and 1 new mutation 1,717-2(A > G).

Detection of the novel cell adhesion molecule MUC18 in human brain tissue.

The implication of cell adhesion molecules (CAMs) in the mediation of the immune response to inflammatory stimuli has been shown in different neurological syndromes. The neural cell adhesion molecule (NCAM) and the myelin associated glycoprotein (MAG) are functionally important CNS-cell adhesion molecules (CAMs) and members of the immunoglobulin gene superfamily (IgSF). Both CAMs are expressed from the time of neuronal induction and myelin formation and are likely to contribute to the generally adhesive properties of neurons and oligodendrocytes. The present study describes, by means of SDS-PAGE and immunoblotting, a novel CNS antigen which is a glycoprotein detected in post mortem frontal lobe tissue of ten subjects at an M(r) 65 and 113 kD which showed the greatest sequence homology to neuronal IgSF members like NCAM. This expected membrane CAM, with probable functions in CNS cell-cell interactions, had been detected first in primary human melanoma cells, with increasing expression as tumors progress and is expected to be a developmentally regulated CAM in neuroectodermal tissues.