IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12ß2, gp130:gp130, IL-12ß2:IL-12ß2, and IL-12ß2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rß1 and IL-23R/IL-12Rß2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

Classic IL-6R signalling is dispensable for intestinal epithelial proliferation and repair.

Inflammatory bowel disease is characterized by disturbed cytokine signalling in the mucosa. Inhibition of the proinflammatory interleukin (IL)-6 pathway is a promising new therapeutic strategy, but safety concerns arise as IL-6 signalling also contributes to epithelial repair of the intestinal mucosa. To which extent IL-6 classic or trans-signalling contributes to intestinal repair remains elusive. We tested the influence of IL-6 classic signalling on intestinal repair and proliferation. Whereas IL-6 induced STAT3 phosphorylation in the colonic cancer cell lines, primary non-malignant intestinal organoids did not respond to IL-6 classic signalling. Mice deficient in intestinal IL-6R (IL-6RΔIEC mice) did not display increased susceptibility to acute dextran sulfate sodium (DSS)-induced colitis. In the azoxymethane DSS model IL-6RΔIEC mice were not protected from inflammation-induced carcinogenesis but showed comparable tumor load to wild-type mice. These data indicate that classic signalling is not the major pathway to transduce IL-6 stimuli into the intestinal epithelium.

STAT3 activation via interleukin 6 trans-signalling contributes to ileitis in SAMP1/Yit mice.

BACKGROUND AND AIMS: SAMP1/Yit mice spontaneously develops intestinal inflammation. Previously, we demonstrated that the signal transducer and activator of transcription (STAT)-3/suppressor of cytokine signalling (SOCS)-3 pathway is pivotal in human inflammatory bowel disease. In our studies in SAMP1/Yit mice, the aim was to investigate whether STAT3 activation contributes to ileitis and to examine the therapeutic effects of this signal blockade. METHODS: Intestinal expression of phospho-STAT3 in SAMP1/Yit mice and control AKR/J mice was examined by western blotting and immunohistochemistry. SOCS3 and interleukin 6 (IL-6) mRNA were determined by northern blotting and reverse transcription-polymerase chain reaction, respectively. We also examined the effects of intravenously injected hyper-IL-6, an IL-6/soluble IL-6 receptor fusion protein, and of soluble gp130-Fc, a specific inhibitor of soluble IL-6 receptor signalling, on STAT3 phosphorylation and disease severity in SAMP1/Yit mice. RESULTS: Phospho-STAT3 was expressed strongly during the disease course in SAMP1/Yit mice but only transiently in AKR/J mice. Phospho-STAT3 was localised to epithelial and mononuclear cells in the diseased intestine of SAMP1/Yit mice. SOCS3 as well as IL-6 mRNAs were expressed in affected intestine. Administration of hyper-IL-6 caused disease exacerbation and enhancement of STAT3 phosphorylation. In contrast, soluble gp130-Fc administration ameliorated the disease and suppressed STAT3 phosphorylation. CONCLUSION: STAT3 signalling is critical in the development of intestinal inflammation in SAMP1/Yit mice. Blockade of this signalling pathway by soluble gp130-Fc may have therapeutic effects in inflammatory bowel disease.

Interleukin-6 trans-signalling in chronic inflammation and cancer.

Interleukin-6 (IL-6) is a cytokine, which plays an important role in many chronic inflammatory diseases. IL-6 belongs to a family of 10 cytokines, which all act via receptor complexes containing the cytokine receptor subunit gp130. On cells, IL-6 first binds to a specific membrane-bound IL-6R and the complex of IL-6 and IL-6R interacts with gp130 leading to signal initiation. Whereas gp130 is widely expressed throughout the body, the IL-6R is only found on some cells including hepatocytes and some leucocytes. A soluble form of the IL-6R is an agonist capable of transmitting signals through interaction with the gp130 protein. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells, which are unresponsive to IL-6 alone, as they do not express the membrane-bound IL-6R. We have named this process trans-signalling. We provided evidence that a soluble form of the IL-6 family signalling receptor subunit gp130 is the natural inhibitor of IL-6 trans-signalling responses. We showed that in chronic inflammatory diseases such as inflammatory bowel disease, peritonitis, rheumatoid arthritis, asthma as well as in colon cancer, IL-6 trans-signalling is critically involved in the maintenance of the disease state. Moreover, in all these animal models, the progression of the disease can be interrupted by specifically interfering with IL-6 trans-signalling using recombinant-soluble gp130Fc protein. The pathophysiologic mechanisms by which the IL-6/soluble IL-6R complex perpetuates the inflammatory state are discussed.

The step response: a method to characterize mechanisms of renal blood flow autoregulation.

Response of renal vasculature to changes in renal perfusion pressure (RPP) involves mechanisms with different frequency characteristics. Autoregulation of renal blood flow (RBF) is mediated by the rapid myogenic response, by the slower tubuloglomerular feedback (TGF) mechanism, and, possibly, by an even slower third mechanism. To evaluate the individual contribution of these mechanisms to RBF autoregulation, we analyzed the response of RBF to a step increase in RPP. In anesthetized rats, the suprarenal aorta was occluded for 30 s, and then the occlusion was released to induce a step increase in RPP. Three dampened oscillations were observed; their oscillation periods ranged from 9.5 to 13 s, from 34.2 to 38.6 s, and from 100.5 to 132.2 s, respectively. The two faster oscillations correspond with previously reported data on the myogenic mechanism and the TGF. In accordance, after furosemide, the amplitude of the intermediate oscillation was significantly reduced. Inhibition of nitric oxide synthesis by Nomega-nitro-l-arginine methyl ester significantly increased the amplitude of the 10-s oscillation. It is concluded that the parameters of the dampened oscillations induced by the step increase in RPP reflect properties of autoregulatory mechanisms. The oscillation period characterizes the individual mechanism, the dampening is a measure for the stability of the regulation, and the square of the amplitudes characterizes the power of the respective mechanism. In addition to the myogenic response and the TGF, a third rather slow mechanism of RBF autoregulation exists.

Transcription of mutS- and mutL-homologous genes during meiosis in Saccharomyces cerevisiae and identification of a regulatory cis-element for meiotic induction of MSH2.

We have analysed the levels of mRNA transcripts of the mutS- and mutL-homologous genes of the yeast Saccharomyces cerevisiae during the course of meiosis, by quantitative RT-PCR. We found that all mutS homologues (MSH1-6) were induced during meiosis, whereas no evidence for regulation of the mutL homologues (PMS1, MLH1-3) was obtained. Temporal expression patterns indicative of co-regulation were observed for the gene pairs MSH4/MSH5 and MSH2/ SPO11. Sequence comparisons of the 5' flanking regions revealed similar sequence stretches in the respective gene pairs, which may constitute regulatory elements. Similar sequences were also found in the 5' flanking regions of the pairs MSH1/MSH3 and MSH1/MSH6. Upstream of MSH2 three closely spaced sequences similar to UASH elements were found, which - surprisingly are located within the coding region of SPO21. Deletion of these elements resulted in loss of meiotic induction of MSH2. Genetic analysis of homozygous deletion mutants did not reveal any differences from wild type with respect to genetic distance estimates, aberrant segregation, or suppression of homoeologus recombination in an interspecies cross with Saccharomyces paradoxus.

Production of spider silk proteins in tobacco and potato.

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420-3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability-a property that is used to purify the spidroins by a simple and efficient procedure.

MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage.

We have characterized the MPH1 gene from Saccharomyces cerevisiae. mph1 mutants display a spontaneous mutator phenotype. Homologs were found in archaea and in the EST libraries of Drosophila, mouse, and man. Mph1 carries the signature motifs of the DEAH family of helicases. Selected motifs were shown to be necessary for MPH1 function by introducing missense mutations. Possible indirect effects on translation and splicing were excluded by demonstrating nuclear localization of the protein and splicing proficiency of the mutant. A mutation spectrum did not show any conspicuous deviations from wild type except for an underrepresentation of frameshift mutations. The mutator phenotype was dependent on REV3 and RAD6. The mutant was sensitive to MMS, EMS, 4-NQO, and camptothecin, but not to UV light and X rays. Epistasis analyses were carried out with representative mutants from various repair pathways (msh6, mag1, apn1, rad14, rad52, rad6, mms2, and rev3). No epistatic interactions were found, either for the spontaneous mutator phenotype or for MMS, EMS, and 4-NQO sensitivity. mph1 slightly increased the UV sensitivity of mms2, rad6, and rad14 mutants, but no effect on X-ray sensitivity was observed. These data suggest that MPH1 is not part of a hitherto known repair pathway. Possible functions are discussed.

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae.

We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs. Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested. Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants. In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants. G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches. Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction. Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants. The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops.

Dyscalculia and dyslexia after right hemisphere injury in infancy.

OBJECTIVE: To use the findings from neuropsychological evaluation and functional magnetic resonance imaging (fMRI) to assess interhemispheric reorganization of function after early unilateral brain injury. DESIGN AND METHODS: The study focused on one case of early brain injury that resulted in both dyscalculia and dyslexia. Brain injury was studied using both structural and fMRI. Intellectual function was evaluated using the Wechsler Intelligence Scale for Children, Third Edition, while visuospatial skills were assessed using the Block Design subtest of the Wechsler Intelligence Scale for Children, Third Edition, and Judgment of Line Orientation subtest. The Selective Reminding Test and the Recurring Figures Test were used to evaluate memory and orientation; language and speech skills were evaluated using the Boston Naming Test, Controlled Oral Word Association, Gates-MacGinitie Reading Test, and color naming. Various methods were used to study arithmetic skills, including the Wide Range Achievement Test-Revised and the Peabody Individual Achievement Test. The control group for fMRI consisted of nine normal subjects. SETTING: Neuropsychological laboratory in primary care hospital. PATIENT: A 17-year-old boy who had sustained a closed head injury associated with a partially depressed, right parietal skull fracture, and right temporal hemorrhage in a motor vehicle crash at age 7 months (November 9, 1977). Subsequent social behavior was normal, but the patient had difficulty throughout school in mathematics and spelling and was characterized as having a "short attention span." INTERVENTION: None. MAIN OUTCOME MEASURES: Standardized tests of arithmetic and reading supplemented by an assessment of calculation and quantitative skills. While performing calculations, fMRI disclosed predominantly left hemisphere activation involving the frontal and posterior parietal regions, whereas this task produced bilateral activation of the supramarginal gyrus in seven of nine normal subjects. RESULTS: Neuropsychological findings confirmed the presence of dyscalculia and dyslexia despite normal intellectual functioning. Visuospatial skills ranged from the low normal to average level. The fMRI findings were consistent with early interhemispheric transfer of visuospatial skills normally committed to the right parietal area to the left parietal region. The patient's dyscalculia and reading ability raise a question of acquired left parietal dysfunction as a consequence of the competition between verbal and visuospatial functions for left hemisphere representation. CONCLUSION: Interhemispheric reorganization of function may be bidirectional rather than a feature unique to the left hemisphere substrate for language.

The history, epidemiology, and classification of headaches in childhood.

The history of headaches in children, their epidemiology, and their classifications are reviewed. Symptoms, prevalence, school absences, risk factors, and treatments are discussed. More data is needed regarding the epidemiology of tension headaches, headaches observed in the emergency room, and headaches observed in the setting of the primary care practitioner.

Does cesarean delivery prevent cerebral palsy or other neurologic problems of childhood?

OBJECTIVE: To determine whether cesarean delivery can lead to fewer childhood neurologic problems. DATA SOURCES: We reviewed English language articles published between 1969 and 1993, obtained via MEDLINE search of the heading "delivery, abdominal." Bibliographies of book chapters and articles about cerebral palsy and other childhood neurologic disorders were also searched. METHODS OF STUDY SELECTION: We sought articles that dealt with vaginal versus cesarean delivery and the following outcomes: cerebral palsy, abnormal neurologic development, neonatal seizures, and neonatal intraventricular hemorrhage. Ten relevant studies were identified, only four of which were prospective, and only one of which (involving breech births) was a randomized trial. DATA EXTRACTION AND SYNTHESIS: No study found a significant difference in the rates of cerebral palsy, abnormal neurologic development, and neonatal seizures between those children born vaginally or by cesarean. The severity of handicap of infants born with myelomeningocele was less in those delivered via cesarean. Infants born by cesarean had a decreased risk for developing neonatal brachial plexus palsy. Cesarean delivery of mothers with human immunodeficiency virus (HIV) or with genital lesions and no history of herpes may benefit the infant. CONCLUSIONS: Cesarean delivery can reduce the risk of adverse childhood neurologic outcome for those born with myelomeningocele, and may reduce the rate of brachial plexus palsies and neonatal herpes and HIV infections. However, children born by cesarean have no documented reduced risk of other childhood neurologic problems or cerebral palsy.