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In Germany, the incidence of cervical cancer, a disease caused by human papillomaviruses (HPV), is higher than in neighboring European countries. HPV vaccination has been recommended for girls since 2007. However, it continues to be significantly less well received than other childhood vaccines, so its potential for cancer prevention is not fully realized. To find new starting points for improving vaccination rates, we analyzed pseudonymized routine billing data from statutory health insurers in the PRÄZIS study (prevention of cervical carcinoma and its precursors in women in Saarland) in the federal state Saarland serving as a model region. We show that lowering the HPV vaccination age to 9 years led to more completed HPV vaccinations already in 2015. Since then, HPV vaccination rates and the proportion of 9- to 11-year-old girls among HPV-vaccinated females have steadily increased. However, HPV vaccination rates among 15-year-old girls in Saarland remained well below 50% in 2019. Pediatricians vaccinated the most girls overall, with a particularly high proportion at the recommended vaccination age of 9-14 years, while gynecologists provided more HPV catch-up vaccinations among 15-17-year-old girls, and general practitioners compensated for HPV vaccination in Saarland communities with fewer pediatricians or gynecologists. We also provide evidence for a significant association between attendance at the children´s medical check-ups "U11" or "J1" and HPV vaccination. In particular, participation in HPV vaccination is high on the day of U11. However, obstacles are that U11 is currently not financed by all statutory health insurers and there is a lack of invitation procedures for both U11 and J1, resulting in significantly lower participation rates than for the earlier U8 or U9 screenings, which are conducted exclusively with invitations and reminders. Based on our data, we propose to restructure U11 and J1 screening in Germany, with mandatory funding for U11 and organized invitations for HPV vaccination at U11 or J1 for both boys and girls., Competing Interests: The authors have declared that no competing interests exist.

Background: Reliable data on the adult SARS-CoV-2 infection fatality rate in Germany are still scarce. We performed a federal state-wide cross-sectional seroprevalence study named SaarCoPS, that is representative for the adult population including elderly individuals and nursing home residents in the Saarland., Methods: Serum was collected from 2940 adults via stationary or mobile teams during the 1 st pandemic wave steady state period. We selected an antibody test system with maximal specificity, also excluding seroreversion effects due to a high longitudinal test performance. For the calculations of infection and fatality rates, we accounted for the delays of seroconversion and death after infection., Results: Using a highly specific total antibody test detecting anti-SARS-CoV-2 responses over more than 180 days, we estimate an adult infection rate of 1.02% (95% CI: [0.64; 1.44]), an underreporting rate of 2.68-fold (95% CI: [1.68; 3.79]) and infection fatality rates of 2.09% (95% CI: (1.48; 3.32]) or 0.36% (95% CI: [0.25; 0.59]) in all adults including elderly individuals, or adults younger than 70 years, respectively., Conclusion: The study highlights the importance of study design and test performance for seroprevalence studies, particularly when seroprevalences are low. Our results provide a valuable baseline for evaluation of future pandemic dynamics and impact of public health measures on virus spread and human health in comparison to neighbouring countries such as Luxembourg or France., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2022.)

Fluorescent in situ hybridization (FISH) provides sensitive detection and visualization of RNA transcripts in tissues and cells with high resolution. We present here a multiplex RNA FISH method using enhanced tyramide signal amplification (TSA) for colocalization analysis of three different transcripts in intact zebrafish brains. To achieve enhancement of fluorescent signals, essential steps of the FISH procedure are optimized including embryo permeability, hybridization efficacy, and fluorogenic TSA-reaction conditions. Critical to this protocol, the enzymatic peroxidase (PO) reactivity is significantly improved by the application of viscosity-increasing polymers, PO accelerators, and highly effective bench-made tyramide substrates. These advancements lead to an optimized TSA-FISH protocol with dramatically increased signal intensity and signal-to-background ratio allowing for visualization of three mRNA transcript patterns simultaneously. The TSA-FISH procedure can be combined with immunofluorescence (IF) to compare mRNA transcript and protein expression patterns.

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Adélaïde Hautval (1906-1988), psychiatrist, was arrested in April 1942 having defended a Jewish family abused by German soldiers. She was imprisoned in Bourges and in several camps in France (Pithiviers, Beaune-la-Rolande, Orléans, Romainville) before being deported as a "Friend of the Jews" at Auschwitz in the convoy of 23 January 1943, said 31,000 convoy with 229 other resistant women, Marie-Claude Vaillant-Couturier, Charlotte Delbo, Danielle Casanova. She refused to participate in the "medical" experience of Nazi doctors Clauberg, Schumann, Wirths and Mengele at Birkenau. She was then deported to Ravensbrück. During her deportation, she illustrated her dedication in the medical management of the deportees. She testified repeatedly on her experience of remote doctor. Hautval Adélaïde was the first French woman doctor named Righteous among the Nations in 1965., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Whole-mount in situ hybridization is the preferred method for detecting transcript distributions in whole embryos, tissues, and organs. We present here a sensitive fluorescent in situ hybridization method for colocalization analysis of different transcripts in whole embryonic zebrafish brains. The method is based on simultaneous hybridization of differently hapten-labeled RNA probes followed by sequential rounds of horseradish peroxidase (POD)-based transcript detection. Sequential detection involves enhancement of fluorescent signals by tyramide signal amplification (TSA) and effective inactivation of the antibody-POD conjugate prior to the following detection round. We provide a detailed description of embryo preparation, hybridization, antibody detection, POD-TSA reaction, and mounting of embryos for imaging. To achieve high signal intensities, we optimized key steps of the method. This includes improvement of embryo permeability by hydrogen peroxide treatment and efficacy of hybridization and TSA-POD reaction by addition of the viscosity-increasing polymer dextran sulfate. The TSA-POD reaction conditions are further optimized by application of substituted phenol compounds as POD accelerators and use of highly efficient bench-made tyramide substrates. The obtained high signal intensities and cellular resolution of our method allows for co-expression analysis and generation of three-dimensional models. Our protocol is tailored to optimally work in zebrafish embryos, but can surely be modified for application in other species as well.

During development of the early neural tube, positional information provided by signaling gradients is translated into a grid of transverse and longitudinal transcription factor expression domains. Transcription factor specification codes defining distinct histogenetic domains within this grid are evolutionarily conserved across vertebrates and may reflect an underlying common vertebrate bauplan. When compared to the rich body of comparative gene expression studies of tetrapods, there is considerably less comparative data available for teleost fish. We used sensitive multicolor fluorescent in situ hybridization to generate a detailed map of regulatory gene expression domains in the embryonic zebrafish diencephalon. The high resolution of this technique allowed us to resolve abutting and overlapping gene expression of different transcripts. We found that the relative topography of gene expression patterns in zebrafish was highly similar to those of orthologous genes in tetrapods and consistent with a three-prosomere organization of the alar and basal diencephalon. Our analysis further demonstrated a conservation of intraprosomeric subdivisions within prosomeres 1, 2, and 3 (p1, p2, and p3). A tripartition of zebrafish p1 was identified reminiscent of precommissural (PcP), juxtacommissural (JcP), and commissural (CoP) pretectal domains of tetrapods. The constructed detailed diencephalic transcription factor gene expression map further identified molecularly distinct thalamic and prethalamic rostral and caudal domains and a prethalamic eminence histogenetic domain in zebrafish. Our comparative gene expression analysis conformed with the idea of a common bauplan for the diencephalon of anamniote and amniote vertebrates from fish to mammals., (Copyright © 2012 Wiley Periodicals, Inc.)

Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.

Background: Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round., Results: We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated., Conclusion: Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Background: In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD) activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected., Results: We developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA). Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH) experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts in different colors in whole-mount zebrafish embryos., Conclusions: Development of a multicolor FISH procedure allowed the comparison of transcript gene expression domains in the embryonic zebrafish brain to a cellular level. Likewise, this method should be applicable for mRNA colocalization studies in any other tissues or organs. The key optimization steps of this method for use in zebrafish can easily be implemented in whole-mount FISH protocols of other organisms. Moreover, our improved reaction conditions may be beneficial in any application that relies on a TSA/POD-mediated detection system, such as immunocytochemical or immunohistochemical methods., (© 2011 Lauter et al; licensee BioMed Central Ltd.)

In this review we address the main cutaneous manifestations and diseases associated with deficiencies in components of the complement system. The first part is devoted to hereditary angioedema, in which acute, sometimes life-threatening recurrent attacks of acute swelling, usually associated with gastrointestinal symptoms, occur. It is related to a structural or functional deficiency of C1 esterase inhibitor. Patients usually have lowered C4 levels, and diagnosis relies on determination of antigenic and/or functional C1 inhibitor level. The second part focuses on lupus erythematosus, as deficiencies in early components of the complement system, such as C1q, C1r, C1s, C2 or C4, are the strongest known disease susceptibility genes for the development of human systemic lupus erythematosus. Severe infections early in life and marked photosensitivity in a patient with lupus erythematosus are clues to an underlying complement deficiency. The genetic background and the clinical associations of the different components of the complement system will be detailed.

The corticotropin-releasing hormone (CRH) family consists of four paralogous genes, CRH and urocortins (UCNs) 1, 2, and 3. In a previous study, we analyzed CRH in the teleost model organism zebrafish and its transcript distribution in the embryonic brain. Here, we describe full-length cDNAs encoding urotensin 1 (UTS1), the teleost UCN1 ortholog, and UCN3 of zebrafish. Major expression sites of uts1 in adult zebrafish are the caudal neurosecretory system and brain. By using RT-PCR analysis, we show that uts1 mRNA is also present in ovary, maternally contributed to the embryo, and expressed throughout embryonic development. Expression of ucn3 mRNA was detected in a range of adult tissues and during developmental stages from 24 hours post fertilization onward. Analysis of spatial transcript distributions by whole-mount in situ hybridization revealed limited forebrain expression of uts1 and ucn3 during early development. Small numbers of uts1-synthesizing neurons were found in subpallium, hypothalamus, and posterior diencephalon, whereas ucn3-positive cells were restricted to telencephalon and retina. The brainstem was the main site of uts1 and ucn3 synthesis in the embryonic brain. uts1 Expression was confined to the midbrain tegmentum; distinct hindbrain cell groups, including locus coeruleus and Mauthner neurons; and the spinal cord. ucn3 Expression was localized to the optic tectum, serotonergic raphe, and distinct rhombomeric cell clusters. The prominent expression of uts1 and ucn3 in brainstem is consistent with proposed roles of CRH-related peptides in stress-induced modulation of locomotor activity through monoaminergic brainstem neuromodulatory systems., ((c) 2010 Wiley-Liss, Inc.)

Mechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive. Here, we have developed a zebrafish tumor model that allows us to study the role of pathological angiogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level. Under normoxia, implantation of a murine T241 fibrosarcoma into the perivitelline cavity of developing embryos of transgenic fli1:EGFP zebrafish did not result in significant dissemination, invasion, and metastasis. In marked contrast, under hypoxia substantial tumor cells disseminated from primary sites, invaded into neighboring tissues, and metastasized to distal parts of the fish body. Similarly, expression of the hypoxia-regulated angiogenic factor, vascular endothelial growth factor (VEGF) to a high level resulted in tumor cell dissemination and metastasis, which correlated with increased tumor neovascularization. Inhibition of VEGF receptor signaling pathways by sunitinib or VEGFR2 morpholinos virtually completely ablated VEGF-induced tumor cell dissemination and metastasis. To the best of our knowledge, hypoxia- and VEGF-induced pathological angiogenesis in promoting tumor dissemination, invasion, and metastasis has not been described perviously at the single cell level. Our findings also shed light on molecular mechanisms of beneficial effects of clinically available anti-VEGF drugs for cancer therapy.

The blood and lymphatic vasculatures are structurally and functionally coupled in controlling tissue perfusion, extracellular interstitial fluids, and immune surveillance. Little is known, however, about the molecular mechanisms that underlie the regulation of bloodlymphatic vessel connections and lymphatic perfusion. Here we show in the adult zebrafish and glass catfish (Kryptopterus bicirrhis) that blood-lymphatic conduits directly connect arterial vessels to the lymphatic system. Under hypoxic conditions, arterial-lymphatic conduits (ALCs) became highly dilated and linearized by NO-induced vascular relaxation, which led to blood perfusion into the lymphatic system. NO blockage almost completely abrogated hypoxia-induced ALC relaxation and lymphatic perfusion. These findings uncover mechanisms underlying hypoxia-induced oxygen compensation by perfusion of existing lymphatics in fish. Our results might also imply that the hypoxia-induced NO pathway contributes to development of progression of pathologies, including promotion of lymphatic metastasis by modulating arterial-lymphatic conduits, in the mammalian system.

Serotonin (5-HT) plays an important role in shaping the activity of the spinal networks underlying locomotion in many vertebrate preparations. At larval stages in zebrafish, 5-HT does not change the frequency of spontaneous swimming; and it only decreases the quiescent period between consecutive swimming episodes. However, it is not known whether 5-HT exerts similar actions on the locomotor network at later developmental stages. For this, the effect of 5-HT on the fictive locomotor pattern of juvenile and adult zebrafish was analyzed. Bath-application of 5-HT (1-20 mum) reduced the frequency of the NMDA-induced locomotor rhythm. Blocking removal from the synaptic cleft with the reuptake inhibitor citalopram had similar effects, suggesting that endogenous serotonin is modulating the locomotor pattern. One target for this modulation was the mid-cycle inhibition during locomotion because the IPSPs recorded in spinal neurons during the hyperpolarized phase were increased both in amplitude and occurrence by 5-HT. Similar results were obtained for IPSCs recorded in spinal neurons clamped at the reversal potential of excitatory currents (0 mV). 5-HT also slows down the rising phase of the excitatory drive recorded in spinal cord neurons when glycinergic inhibition is blocked. These results suggest that the decrease in the locomotor burst frequency induced by 5-HT is mediated by a potentiation of mid-cycle inhibition combined with a delayed onset of the subsequent depolarization.

Complete deficiency of complement C4 is among the strongest genetic risk factors for human systemic lupus erythematosus (SLE). C4 is a constituent of the RP-C4-CYP21-TNX (RCCX) module in the human leukocyte antigen (HLA) that exhibits inter-individual copy-number and gene-size variations. Here, we studied two North-African families with complete C4 deficiency and SLE. The first included a Moroccan male SLE patient (1P) and a sibling, who were both homozygous for HLA-A*02 B*17 DRB1*07. The second had an Algerian female SLE patient (2P) homozygous for HLA-A*01 B*17 DRB1*13. Early SLE disease onset, the presence of photosensitive rashes, anti-Ro/SSA, renal disease and high titers of antinuclear antibodies were the common features of complete C4 deficiency. Southern blot analyses showed that 1P had monomodular RCCX with a long C4A, whereas 2P had bimodular RCCX with one long C4A and one short C4B. Genomic DNA fragments for these mutant genes were amplified and sequenced. A C>T transition that created the R540X nonsense mutation in C4A was found in 1P. An identical 4-bp insertion that generated the Y1537X nonsense mutation was discovered in both C4A and C4B of 2P. The high concordance of SLE and C4 deficiency among patients with non-DR3 and non-DR2 haplotypes underscores the importance of C4 proteins in the protection against SLE.

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs.

Mammalian liver-X-receptors (LXRs) are transcription factors activated by oxysterols. They play an essential role in lipid and glucose metabolism. We have cloned the open reading frame of zebrafish lxr and describe its genomic organization. Zebrafish lxr encodes a 50-kDa protein with high sequence similarity to mammalian LXRalpha. In transfection assays, the encoded protein showed transcriptional activity in response to LXR-ligands. Treatment of adult zebrafish with the synthetic LXR ligand, GW3965, induced expression of genes involved in hepatic cholesterol and lipid pathways. Using qPCR and in situ hybridization, we found ubiquitous expression of lxr mRNA during the first 24 hr of development, followed by more restricted expression, particularly to the liver at 3dpf and the liver and intestine at 4dpf. In adult fish, all examined organs expressed lxr. In addition to a metabolic role of lxr, the temporal expression pattern suggests a developmental role in, e.g., the liver and CNS., ((c) 2008 Wiley-Liss, Inc.)

The zebrafish is an attractive model system for studying the function of the spinal locomotor network by combining electrophysiological, imaging, and genetic approaches. Thus far, most studies have been focusing on embryonic and larval stages. In this study we have developed an in vitro preparation of the isolated spinal cord from adult zebrafish in which locomotor activity can be induced while the activity of single neurons can be monitored using whole cell recording techniques. Application of NMDA elicited rhythmic locomotor activity that was monitored by recording from muscles or ventral roots in semi-intact or isolated spinal cord preparations, respectively. This rhythmic activity displayed a left-right alternation and a rostrocaudal delay. Blockade of glycinergic synaptic transmission by strychnine switched the alternating activity into synchronous bursting in the left and right sides as well as along the rostrocaudal axis. Whole cell recordings from motoneurons showed that they receive phasic synaptic inputs that were correlated with the locomotor activity recorded in ventral roots. This newly developed in vitro preparation of the adult zebrafish spinal cord will allow examination of the organization of the spinal locomotor network in an adult system to complement studies in zebrafish larvae and new born rodents.

Corticotropin-releasing hormone (CRH) plays a central role in the physiological regulation of the hypothalamus-pituitary-adrenal/interrenal axis mediating endocrine, behavioral, autonomic, and immune responses to stress. Despite the wealth of knowledge about the physiological roles of CRH, the genetic mechanisms by which CRH neurons arise during development are poorly understood. As a first step toward analyzing the molecular and genetic pathways involved in CRH lineage specification, we describe the developmental distribution of CRH neurons in the embryonic zebrafish, a model organism for functional genomics and developmental biology. We searched available zebrafish expressed sequence tag (EST) databases for CRH-like sequences and identified one EST that contained the complete zebrafish CRH open reading frame (ORF). The CRH precursor sequence contained a signal peptide, the CRH peptide, and a cryptic peptide with a conserved sequence motif. RT-PCR analysis showed crh expression in a wide range of adult tissues as well as during embryonic and larval stages. By whole-mount in situ hybridization histochemistry, discrete crh-expressing cell clusters were found in different parts of the embryonic zebrafish brain, including telencephalon, preoptic region, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, and rostral hindbrain and in the neural retina. The localization of crh mRNA within the preoptic region is consistent with the central role of CRH in the teleost stress response through activation of the hypothalamic-pituitary-interrenal axis. The widespread distribution of CRH-synthesizing cells outside the preoptic region suggests additional functions of CRH in the embryonic zebrafish brain., ((c) 2007 Wiley-Liss, Inc.)

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain-hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.

Introduction: Deficiencies in components of the classical pathway of complement activation are strong risk factors for lupus erythematosus (LE).Yet, it has not been addressed whether the conventional measurements of the serum hemolytic CH50 activity and antigenic concentrations of C3 and C4 are sufficient to asses a deficiency in C4A, C4B or C2 components, the most common deficiencies associated with LE., Patients and Methods: In a retrospective series, we performed complement analyses in 35 patients with LE who were systematically screened for a complement deficiency. The majority of patients had cutaneous LE with mild systemic involvement and no complement consumption. Of 25 patients (72%) with complement deficiency we found 13 with a partial C4A deficiency, 2 with a complete C4A deficiency, 6 with a partial C4B deficiency, 2 with a complete C4B deficiency and 2 with a combined partial C2 and C4A deficiency., Results: The total complement activity (CH50) was decreased in only one out of two patients with complete C4B deficiency. CH50 level was found to be low-normal (35-38 U/ml(-1)) in one patient with partial C4B deficiency, one patient with complete C4B deficiency and both patients with combined partial C4A and C2 deficiency. Total C4 levels were normal in 9 out of 13 the patients with a partial C4A deficiency and in 2 out of 6 patients with a complete C4B deficiency. The antigenic concentration of C3 was low in only 1 patients with a complete C4B deficiency and within the normal range in all the others patients. Overall, 50% of the patients had normal or elevated C3, C4, and CH50 levels., Discussion: This study emphasizes that the usual measurements of CH50, C3 and C4 levels are not adequate to detect a C4 and/or C2 deficiency in patients with LE. In epidemiologic or investigative studies addressing the prevalence of complement deficiency, more elaborated diagnostic tests, such as C4 protein allotyping, C2 level measurement and genetic screening for type I C2 deficiency should also be performed.

Background: Although deficiencies in the early components of the complement system were among the first identified genetic risk factors for systemic lupus erythematosus (SLE), only a few studies addressed their significance in patients with cutaneous LE (CLE). Among environmental factors, it was postulated that cigarette smoking might intervene in the pathogenesis of LE., Objectives: To describe the clinical and biological features of patients with CLE and a complement deficiency. A secondary objective was to assess cigarette smoking in patients with CLE., Patients and Methods: A retrospective study including all patients diagnosed as having LE between 1995 and 2003 in the Dermatology Department of Strasbourg University Hospital. Patient charts were reviewed and those patients in whom a C4 and/or C2 deficiency was diagnosed were included. Two patients with a combined C2/C4 deficiency were analysed in detail., Results: There were 48 females and 37 males (F/M ratio = 1.3), with a mean age of 41 years at diagnosis; 73% of the patients had chronic LE and 27% subacute CLE. Among 32 screened patients, 24 patients with a mean age of 36 years had a complement deficiency; 17 had a C4A deficiency, five a C4B deficiency and two a combined C4A/C2 deficiency. A high proportion (58%) of these patients was male; 82% of the patients were smokers. This was especially true in males: 94% were smokers compared with 69% of females., Conclusions: Partial deficiency of C4, C2 or C4 and C2 is a common finding in patients with CLE. Most male patients with CLE are smokers. It is thus suggested that the combination of cigarette smoking and complement deficiency could be a risk factor for LE in men.

The MHC, primarily known for its antigen-presenting class I and II molecules, harbours, within a central segment of less than 1 Mb, a dense collection of genes involved in various biological functions. Although MHC I and MHC II are principal players of adaptive immunity, several loci within this central (still called class III) MHC region encode members of the innate immune system. These include the long known factors of the complement system--potentially inhibitory and triggering natural killer receptors as well as stress proteins. Whether this physical proximity is fortuitous or functionally advantageous is an important question for the future of MHC genetics.

CNS catecholaminergic neurons can be distinguished by their neurotransmitters as dopaminergic or noradrenergic and form in distinct regions at characteristic embryonic stages. This raises the question of whether all catecholaminergic neurons of one transmitter type are specified by the same set of factors. Therefore, we performed genetic analyses to define signaling requirements for the specification of distinct clusters of catecholaminergic neurons in zebrafish. In mutants affecting midbrain- hindbrain boundary (MHB) organizer formation, the earliest ventral diencephalic dopaminergic neurons appear normal. However, after 2 d of development, we observed fewer cells than in wild types, which suggests that the MHB provides proliferation or survival factors rather than specifying ventral diencephalic dopaminergic clusters. In hedgehog (Hh) pathway mutants, the formation of catecholaminergic neurons is affected only in the pretectal cluster. Surprisingly, neither fibroblast growth factor 8 (FGF8) alone nor in combination with Hh signaling is required for specification of early developing dopaminergic neurons. We analyzed the formation of prosomeric territories in the forebrain of Hh and Nodal pathway mutants to determine whether the absence of specific dopaminergic clusters may be caused by early patterning defects ablating corresponding parts of the CNS. In Nodal pathway mutants, ventral diencephalic and pretectal catecholaminergic neurons fail to develop, whereas both anatomical structures form at least in part. This suggests that Nodal signaling is required for catecholaminergic neuron specification. In summary, our results do not support the previously suggested dominant roles for sonic hedgehog and Fgf8 in specification of the first catecholaminergic neurons, but instead indicate a novel role for Nodal signaling in this process.

Reduction of mortality is a basic goal of methadone maintenance treatment (MMT) for opioid addicts. During the last years, however, there have been reports about drug related deaths of MMT patients and of untreated drug addicts who had consumed illegally purchased methadone. To evaluate the supposed beneficial effect of MMT on mortality, data from a multicentre trial in the German state of North Rhine-Westphalia were analysed. 244 patients with long histories of opioid addiction and previous failures in at least two abstinence oriented treatments were observed for at least four years and up to eight years after admission to MMT. With 32 deaths out of 244 participants, the mortality rate for the whole sample was 2.5 % p.a. This rate is not remarkably different from estimations for opioid addicts in general. However, addicts in continuous maintenance treatment had a mortality rate of only 1.6 % p.a., in contrast to a rate of 8.1 % p.a. for addicts who had left treatment. These results are supported by other long-term studies with large patient samples. Therefore it is concluded that methadone maintenance treatment is a measure to reduce the mortality in opioid addicts. Attainment of this goal is threatened, however, by deviations from safety standards, especially if take-home privileges are granted too widely.

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.

During early developmental stages, the embryonic vertebrate brain is still relatively simple with few morphological landmarks that would indicate subdivisions in the prosencephalic primordium. To better understand the early organization of the rostral brain of a lower vertebrate, we investigated the embryonic development and regionalization of the fore- and midbrain of a small teleost, the zebrafish (Danio rerio). We used regulatory gene expression patterns to trace putative prosomeric domains to the beginning of the pharyngula period, when morphological manifestations of prosomeres are not immediately evident. We directly compared the expression domains of members of the dlx, emx, fgf, hh, lim, nkx, otx, pax, POU, winged helix and wnt regulatory gene families in the rostral brain by means of two-color whole-mount in situ hybridization. This allowed us to define precisely abutting expression borders of neighboring expression domains of different genes. Our analysis shows that the genes examined are expressed in anteroposteriorly and dorsoventrally restricted domains, and share expression borders at stereotypic positions within the fore- and midbrain. The arrangement of the various expression domains identified four major longitudinal subdivisions, which extend in parallel to the bent longitudinal rostral brain axis. Furthermore, we identified a series of eight transverse diencephalic domains which may indicate a prosomeric organization of the rostral zebrafish brain.

The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and neuronal differentiation in the midbrain and anterior hindbrain. Formation of this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and maintenance of its regional identity and signaling activities. Juxtaposition of the Otx2 and Gbx2 expression domains positions the MHB. How the positional information is translated into activation of Pax2, Wnt1 and Fgf8 expression during MHB establishment remains unclear. In zebrafish spiel ohne grenzen (spg) mutants, the MHB is not established, neither isthmus nor cerebellum form, the midbrain is reduced in size and patterning abnormalities develop within the hindbrain. In spg mutants, despite apparently normal expression of otx2, gbx1 and fgf8 during late gastrula stages, the initial expression of pax2.1, wnt1 and eng2, as well as later expression of fgf8 in the MHB primordium are reduced. We show that spg mutants have lesions in pou2, which encodes a POU-domain transcription factor. Maternal pou2 transcripts are distributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection of pou2 mRNA can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 mutants without inducing ectopic expression. This indicates an essential but permissive role for pou2 during MHB establishment. pou2 is expressed normally in noi/pax2.1 and ace/fgf8 zebrafish mutants, which also form no MHB. Thus, expression of pou2 does not depend on fgf8 and pax2.1. Our data suggest that pou2 is required for the establishment of the normal expression domains of wnt1 and pax2.1 in the MHB primordium.

This article contains detailed protocols for the localization of mRNA transcripts within whole Drosophila embryos. The procedures are based on the use of digoxigenin-, fluorescein-, and biotin-labeled antisense RNA probes for nonradioactive detection of transcripts. The labels are visualized in situ by differently colored water-insoluble precipitates using alkaline phosphatase- or beta-galactosidase-based immunoassays. First, a basic method is described that allows detection of transcript distribution(s) of one or more genes using the same color precipitate. Second, a sequential alkaline phosphatase detection method is presented that permits the visualization of two or three independent transcript patterns in multiple colors in the same embryo. Third, a shortened two-color in situ hybridization protocol is provided that employs a combination of beta-galactosidase and alkaline phosphatase colorimetric reactions for differential detection. The two-color in situ hybridization methods work equally well in Drosophila and zebrafish embryos and may therefore also be adaptable to other species., (Copyright 2001 Academic Press.)

Background: The gene deletion responsible for the type I human complement C2 deficiency was reported in 1992. The purpose of our study is to evaluate clinical and immunological characteristics of 11 patients with lupus erythematosus and type I C2 deficiency., Observations: We observed 5 patients with a homozygous C2 deficiency and 6 with a heterozygous C2 deficiency. Eight patients had systemic lupus erythematosus, 2 had subacute cutaneous lupus erythematosus, and 1 had chronic lupus erythematosus. Photosensitivity was present in 73% of the patients, and 64% tested positive for anti-Ro (SSA) antibodies. Renal involvement that required immunosuppressive therapy was present in 54% of the patients. Ninety percent of the patients tested positive for antinuclear antibodies, and 54% tested positive for anti-double-stranded DNA antibodies. Phenotyping of the fourth component of the complement was performed in 82% of the patients and showed a C4A4B2 phenotype, which is suggestive for the type I C2 deficiency., Conclusions: Most patients with lupus erythematosus associated with C2 type I deficiency are photosensitive, and this is probably related to the presence of anti-Ro (SSA) autoantibodies. The prognosis for those patients is not better than that for patients with lupus erythematosus in general.

Vertebrate class III POU genes are widely expressed in the embryonic and adult central nervous system, where they act as transcriptional regulators of cell- and/or region-specific gene expression. We isolated four zebrafish class III POU genes, named zp-12, zp-23, zp-47 and zp-50. In this study, we examined the developmental expression patterns of the Brn-1- and Brn-2-related zp-12, zp-23 and zp-47 genes by means of whole-mount in situ hybridization. Similarly to their mammalian orthologues, the major expression site of all zebrafish zp genes is the CNS. Neurectodermal expression was first detected at the beginning of somitogenesis in spatially restricted segment-like domains in different parts of the neural plate. During somitogenesis transcript distributions changed from highly restricted to widespread but nevertheless distinct patterns found in all major subdivisions of the CNS. While zp-47 expression was detected exclusively in the CNS, localized expression of zp-12 and zp-23 was also found in the pronephric primordium and in cell clusters within the mandibular and hyoid arches. Furthermore, zp-23 transcripts were transiently detected in a restricted region of the paraxial mesendoderm and, at late embryogenesis stages, in the auditory vesicles. The early regionalized expression of all three zp genes is compatible with roles in regional specification of the neural plate. Comparison of the distinct yet overlapping expression of zp-12, zp-23, zp-47 and the previously characterized zp-50 gene implies both unique, as well as redundant functions for each family member. We propose that coordinate expression of particular combinations of class III POU genes contribute to pattern formation or cell fate determination in the developing CNS and other structures., (Copyright 2000 Wiley-Liss, Inc.)

To shed light on the organization of the rostral embryonic brain of a lower vertebrate, we have directly compared the expression patterns of dlx, fgf, hh, hlx, otx, pax, POU, winged helix and wnt gene family members in the fore- and midbrain of the zebrafish. We show that the analyzed genes are expressed in distinct transverse and longitudinal domains and share expression boundaries at stereotypic positions within the fore- and midbrain. Some of these shared expression boundaries coincide with morphological landmarks like the pathways of primary axon tracts. We identified a series of eight transverse diencephalic domains suggestive of neuromeric subdivisions within the rostral brain. In addition, we identified four molecularly distinct longitudinal subdivisions and provide evidence for a strong bending of the longitudinal rostral brain axis at the cephalic flexure. Our data suggest a strong conservation of early forebrain organization between lower and higher vertebrates.

Properdin deficiency was demonstrated in three generations of a large Swiss family. The concentration of circulating properdin in affected males was < 0.1 mg/l, indicating properdin deficiency type I. Two of the nine properdin-deficient males in the family had survived meningitis caused by Neisseria meningitidis serogroup B without sequel. Two point mutations were identified when the properdin gene in one of the properdin-deficient individuals was investigated by direct solid-phase sequencing of overlapping polymerase chain reaction (PCR) products. The critical mutation was found at base 2061 in exon 4, where the change of cytosine to thymine had generated the stop codon TGA. The other mutation was positioned at base 827 in intron 3. The stop codon in exon 4 was also demonstrated by standard dideoxy sequencing in three additional family members. The question was asked if genetic factors such as partial C4 deficiency and IgG allotypes could have influenced susceptibility to meningococcal disease in the family. No relationship was found between C4 phenotypes and infection. Interestingly, the two properdin-deficient males with meningitis differed from the other properdin-deficient persons in that they lacked the G2m(n) allotype, a marker known to be associated with poor antibody responses to T-independent antigens. This implies that the consequences of properdin deficiency might partly be determined by independent factors influencing the immune response.

A fast and simplified two-color in situ hybridization procedure for the simultaneous detection of two different mRNAs in whole-mounted zebrafish and Drosophila embryos is presented. Transcript distributions are detected in a single incubation step using a mixture of alkaline phosphatase and beta-galactosidase coupled antibodies. The different transcripts are visualized in contrasting colors by the use of beta-galactosidase substrates that develop color precipitates (magenta, blue) easily distinguishable from those of the standard alkaline phosphatase substrates. This protocol can be followed by standard immunohistochemistry to detect the expression of a third gene (at the protein level) in a third color.

Background & Aims: Despite extensive investigations of portal vein thrombosis, no underlying cause is identifiable in up to 30% of patients. A recently described mutation of the prothrombin gene at nucleotide position 20210 is associated with history of venous thrombosis and was assessed in this study., Methods: We compared the frequency of factor II G20210A and factor V G1691A (factor V Leiden) mutations in 10 patients with idiopathic portal vein thrombosis, 10 patients with nonidiopathic portal vein thrombosis, 60 patients with deep vein thrombosis of the legs, and 42 control subjects., Results: The frequency of factor II G20210A mutation was increased in patients with idiopathic portal vein thrombosis (40.0%; confidence interval, 3.1%-76.9%) compared with controls (4.8%; confidence interval, 0%-11.5%) or patients with nonidiopathic portal vein thrombosis or deep vein thrombosis (P = 0.0001). In contrast, the frequency of the factor V G1691A mutation was similar in subjects with portal vein thrombosis and in controls but was increased in patients with deep vein thrombosis (P = 0.0001)., Conclusions: The factor II G20210A mutation is frequent in patients with idiopathic portal vein thrombosis and should therefore be assessed under this circumstance.

Objective: To study the variations of type II soluble receptors for tumor necrosis factor (sR-TNF) in patients with systemic lupus erythematosus and investigate their use in the clinical setting., Patients and Methods: Twenty-six patients with systemic lupus were followed for a mean 3 years. sR-TNF and other immunological parameters (C reactive protein, anti-DNA antibodies, C3 and C4 complement fractions, soluble receptors for interleukin 2) were measured in sera at different points of the disease course. The systemic lupus activity measure (SLAM) was determined at each point, and confronted with the biology results. The study was cross sectional for the group and longitudinal for the patients., Results: sR-TNF was the immunological parameter which correlated best with SLAM. It also correlated with sedimentation rate, C reactive protein, thrombopenia, anemia, creatinine level, anti-DNA antibodies and sR-IL2. The longitudinal study pointed out however that this finding is not consistent for each patient., Conclusion: A rise in sR-TNF related to systemic lupus activity but is of limited practical interest for individual patient follow-up.

Background: This investigation describes the preclinical development of a laser fiberoptic interstitial delivery system for the thermal destruction of small breast cancers. We propose adaptation of this technology to stereotactic mammographic instrumentation currently employed for diagnostic core biopsy to thermally ablate a site of disease with maximal treatment efficacy, minimal observable superficial change, reduced patient trauma, and lowered overall treatment costs., Study Design: Laser hyperthermia is a clinical modality that seeks to achieve tumor destruction through controlled tissue heating. The advantage of laser-induced hyperthermia over traditionally used heat sources such as ultrasound, microwave, or radiowave radiation lies in the ability to focus heat localization to the specific tumor tissue site. Neodymium:yttrium aluminum garnet (Nd:YAG) laser light transmitted through a fiberoptic cable to a diffusing quartz tip can induce such temperature increases leading to localized tissue destruction. Because breast cancer occurs with greatest frequency in the mature woman whose breast tissue has undergone glandular involution with fatty replacement, this study concentrates on determining the resultant laser energy heat distribution within fat and fibrofatty tissue. This investigation studied the time-temperature responses of ex vivo human breast and porcine fibrofatty tissue, which led to an in vivo subcutaneous porcine model for the practical demonstration of a laser hyperthermia treatment of small volumes of porcine mammary chain tissue., Results: Spatial recordings of the resultant temperature fields through time exhibited similar, reproducible thermal profiles in both ex vivo human breast and subcutaneous porcine fat. In vivo laser-produced temperature fields in porcine subcutaneous fat were comparable to those in the ex vivo analyses, and showed a histologically, sharply defined, and controllable volume of necrosis with no injury to adjacent tissues or to overlying skin., Conclusions: Interstitially placed, fiberoptically delivered Nd:YAG laser energy is capable of controlled tissue denaturation to a defined volume for the treatment of small breast cancers. It is hoped that this minimally invasive approach, with further investigation and refinement, may lead to the effective treatment of small, well-defined breast cancers that are commonly diagnosed through stereographic mammography and stereotactic core biopsy. The juxtaposition of such a localized treatment modality with these increasingly used diagnostic tools is of considerable promise.

Purpose: Administration of antithyroid agents, surgery and radioiodine therapy are the established methods for treating hyperthyroidism. All of the methods are relatively complex and are associated with risks. That is why the sonographically controlled minimum invasive alcohol injection method was developed. The disadvantage associated with this procedure, however, is that it can not be controlled as effectively., Methods: Thyroid gland tissue is removed from an animal model with delimitation by means of thermal coagulation using a new, specially developed ITT laser probe. The treatment is effected by puncturing intact skin. Placing of the probe is controlled sonographically. The puncture channel is 18 G; the laser probe has a diameter of 0.8 mm. The procedure is effected using local anesthesia which may be combined with light analgesic sedation and takes approx. 10 minutes. The laser application lasts 145 seconds and has a total power of 1195 joules., Results: Laser treatment can be compared with the invasiveness of a jugular vein catheter unit and has good tolerance. Complete histologic healing of the coagulation zone takes 4 weeks and the boundaries of the scar contraction are barely recognizable. The size of the coagulation zone can only be controlled to a certain degree in the animal model on account of its anatomy., Conclusions: The minimal invasive method based on laser-induced reduction of thyroid gland tissue can be performed on an animal model and is safe. Further research needs to be done to determine the extent to which the results can be transferred to human beings.

Analysis of an abnormal C1q molecule of individuals of a Moroccan family by ultracentrifugation in sucrose gradients revealed a low molecular weight C1q (LMW-C1q). We investigated the molecular basis of this defect by sequencing all six exons of the three C1q genes. One point mutation in the codon for Gly at position 15 (GGT) of the B chain was found resulting in an amino acid substitution to Asp (GAT). The exchange not only leads to an interruption of the collagen-like motif Gly-X-Y, but also introduces one negatively charged residue per B chain which results in two additional charges per structural subunit (A-B, C-C, A-B). The mutation which has been identified by DNA-sequencing in the C1q-deficient younger brother of the propositus was confirmed by PCR-EcoRV-RFLP in the sister and the propositus himself. This mutation is very similar to a mutation previously described in another case of functional C1q deficiency where Gly at position 6 of the C chain was substituted by a large positively charged residue (Arg). Again, a LMW-C1q was demonstrated. These point mutations that lead to amino acid substitutions result in the production of a LMW-C1q where the formation of functionally active 11S C1q consisting of three structural subunits appears to be inhibited by the introduction of six additional charges, one per B or C chain, respectively, in the collagenous region of the molecule.

Intramural temperatures were monitored during catheter-directed endomyocardial non-contact laser irradiation by means of an array of thermocouples inserted epicardially. Via a novel electrode-laser catheter, a total of 30 Nd-YAG laser pulses, 1064 nm, 25 W/15 s, 20 W/30 s, 15 W/45 s,n = 10 each, were aimed at various endocardial sites in the ventricles of four dogs. Temperatures increased gradually after the onset of the laser pulses. Maximum values were measured in the central area of the irradiated spots: 100 +/- 15 double daggerC at 25 W, 78+/- 23 double daggerC at 20 W and 80 +/- 13 double daggerC at 15 W. Application of 300 J of laser energy induced significantly higher temperatures at 25 W (p< 0.02) than at 20 or 15 W (20vs 15 W: p> 0.05). Initial temperature rise and lesion formation was accompanied by dwindling of local electrical potentials. There was no crater formation. Transcatheter non-contact Nd-YAG laser coagulation of healthy ventricular myocardium can be performed in a safe and controllable manner at power settings up to 20 W and laser energies up to 675 J.

The present case report describes a 27-year-old patient who presented with post-traumatic pleural effusion. Analysis of the pleural fluid showed hypereosinophilia (990 mm-3), a decreased level of total complement, and decreased levels of C3 and C4 fractions (less than 50% of normal serum levels), indicating a local consumption mechanism for complement. Complement serum levels (CH50, C3, C4) were normal. All other aetiologic possibilities were eliminated. This case suggests that the immunopathological mechanism of post-traumatic pleural effusion may involve activation of the classical pathway of complement and a recruitment of inflammatory cells such as eosinophils.

Genetic deficiencies of components of the classical pathway of complement activation are associated with an increased risk for the development of autoimmune and immune complex-mediated diseases. In the present study we report on the molecular and clinical features associated with combined heterozygous C4 and C2 deficiency in 15 individuals investigated within six families. Approximately 30% of the individuals manifested SLE or another autoimmune condition. Heterozygous C2 deficiency was related to a 28-bp deletion in the C2 gene (C2 deficiency type I), in most cases within the HLA-A25 B18 C2Q0 BfS C4A4B2 DR2 haplotype. Among 13 partial C4-deficient haplotypes transmitted, 8 carried C4A*Q0 alleles and 5 C4B*Q0 alleles. In seven cases the C4A*Q0 alleles were associated with a deletion of the C4A/CYP21P genes within the HLA-B8 C2C BfS C4AQ0B1 DR3 haplotype. In three cases, the C4B*Q0 allele was associated with a deletion of the C4B/CYP21P genes within the HLA-B18 C2C BfF1 C4A3BQ0 DR3 haplotype. In the other cases, C4A*Q0 or C4B*Q0 was dependent on as yet uncharacterized defects in the C4 gene or in C4 gene expression. In view of the relatively high frequency of heterozygous C4 deficiency in the normal Caucasian population, the expected frequency of the combined deficiency should approximate 0.001.

POU genes encode a family of transcription factors involved in a wide variety of cell fate decisions and in the regulation of differentiation pathways. We have searched for POU genes in the zebrafish, a popular model organism for the study of early development of vertebrates. Besides five putative pseudogenes we have identified five POU genes that are expressed during embryogenesis. Probes obtained by PCR were used to isolate full-length cDNAs. Four of the isolated genes encode proteins with class III POU domains. Analysis of genomic clones suggests that the fish genes in general do not contain introns, similar to class III genes of mammals. However, the C-termini of two of the encoded proteins vary due to facultative splicing of a short intervening sequence. These two genes show very strong similarities in their sequence. They have probably arisen by gene duplication, possibly as part of a larger scale duplication of part of the zebrafish genome. Analysis of the expression of the class III genes shows that they are predominantly expressed in the central nervous system and that they may play important roles in patterning the embryonic brain.