Multimodal Regulation Orchestrates Normal and Complex Disease States in the Retina.

Regulation of biological processes occurs through complex, synergistic mechanisms. In this study, we discovered the synergistic orchestration of multiple mechanisms regulating the normal and diseased state (age related macular degeneration, AMD) in the retina. We uncovered gene networks with overlapping feedback loops that are modulated by nuclear hormone receptors (NHR), miRNAs, and epigenetic factors. We utilized a comprehensive filtering and pathway analysis strategy comparing miRNA and microarray data between three mouse models and human donor eyes (normal and AMD). The mouse models lack key NHRS (Nr2e3, RORA) or epigenetic (Ezh2) factors. Fifty-four total miRNAs were differentially expressed, potentially targeting over 150 genes in 18 major representative networks including angiogenesis, metabolism, and immunity. We identified sixty-eight genes and 5 miRNAS directly regulated by NR2E3 and/or RORA. After a comprehensive analysis, we discovered multimodal regulation by miRNA, NHRs, and epigenetic factors of three miRNAs (miR-466, miR1187, and miR-710) and two genes (Ell2 and Entpd1) that are also associated with AMD. These studies provide insight into the complex, dynamic modulation of gene networks as well as their impact on human disease, and provide novel data for the development of innovative and more effective therapeutics.

Progression or Regression? - Strengths and Weaknesses of the New Munich Nomenclature III for Cervix Cytology.

Introduction: Since 01. 01. 2015 the new Munich nomenclature III for gynaecological diagnostics of the cervix has been in force. The changes have led to controversial scientific discussions. This study reports for the first time on the consequences. Materials and Methods: The present data are based on smear screening results for the year 2014. The data of 63 134 patients were evaluated. Results: 2.27 % of all smears were remarkable. Group IIa was assigned to 0.91 %. Group II-p was somewhat more frequently recorded than group IIID1 (0.59 vs. 0.53 %). Groups IIID1 and IIID2 were found in 0.53 and 0.61 %, respectively, of the cases. Agreement with histology was found in 36.84 and 44.68 %, respectively. Glandular lesions represented the most frequent changes in group III. Histological clarification was obtained for 0.18 % of all remarkable findings. The relative incidence of high-grade precancerous conditions (CIN III) and invasive tumours amounted to 0.1 %. Conclusion: A close communication between gynaecologists and cytologists is mandatory for the correct usage of the new nomenclature. The future annual statistics of the health insurances can now be analysed in more detail. A statistical classification of glandular epithelial changes is now also possible for the first time. The heterogeneous group IIa constitutes an unnecessary uncertainty for patients and physicians. The splitting of the group IIID does not appear to have any advantage for the further clinical management. Further studies are needed to show whether or not the classification can stand up to international comparisons.

Predictors of sustained ventricular arrhythmia episodes in patients with primary ICD indication: male gender and AF in primary ICD prophylaxis.

AIMS: Implantable cardioverter-defibrillators (ICD) reduce mortality in patients with severely impaired left ventricular function. In randomized studies, female patients are underrepresented and data on ICD therapy is limited. Atrial fibrillation (AF) is a determinant of poor prognosis but has not been consistently evaluated. We evaluated the risk factors for the occurrence of ventricular arrhythmia episodes in patients with primary ICD prophylaxis. METHODS: Consecutive patients after ICD implantation for primary prophylaxis were followed. During follow-up, detected sustained episodes of ventricular arrhythmia were documented. Multivariate analysis controlled for propensity score was used to evaluate the correlation between gender, history of AF, and the occurrence of ventricular arrhythmia episodes. RESULTS: A total of 400 patients (19.8% female; n = 79) were included. During follow-up, 64 patients (16%) had appropriate ICD therapy episodes. Men (18%) had significantly more often episodes than women (8%; p = 0.025). Patients with a history of AF (102, 25.5%) had significantly more often episodes (30%) compared to patients without a history of AF (11%; p < 0.001). In a multivariate model, only gender (p = 0.02) and history of AF (p < 0.001) were significantly associated predictors of the occurrence of appropriate ICD therapies during follow-up. Based on the propensity score model, the adjusted hazard ratio for male gender was 2.7 (p = 0.02) and 2.6 (p = 0.0004) for history of AF. CONCLUSION: Male gender and history of AF are independent predictors for the occurrence of sustained ventricular arrhythmia in primary ICD prophylaxis. Further studies need to evaluate whether history of AF in female patients might be an indicator for higher risk of sudden cardiac arrhythmic death.

The production of anti-inflammatory cytokines in whole blood by physico-chemical induction.

OBJECTIVE AND DESIGN: Cytokines such as interleukin-1 (IL-1) play an important role in degenerative musculo-skeletal diseases, including osteoarthritis, and a multitude of inflammatory disorders. Agents that inhibit the action of such cytokines have a high therapeutic potential in such diseases. Here we describe a new method for enhancing the production of the interleukin-l receptor antagonist (IL-1Ra) and other anti-inflammatory cytokines in whole blood. MATERIAL AND METHODS: Human venous blood was incubated in the presence of CrSO(4)-treated glass beads. Serum was recovered and the concentrations of IL-1Ra and other relevant cytokines were measured by ELISA. RESULTS: The interaction of the glass bead surface with cells in whole blood increased production of IL-1Ra and anti-inflammatory cytokines. Removal of the beads and centrifugation generated a serum preparation enriched in anti-inflammatory cytokines. This preparation is of therapeutic value in treating various inflammatory and degenerative disorders. CONCLUSIONS: The increased de novo production of anti-inflammatory cytokines by a direct physico-chemical induction of whole blood in the Orthokin system is feasible and offers an alternative, novel approach to treating mild to moderate OA and other orthopaedic conditions such as degenerative spine diseases.

Expression of two nblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp. PCC6803.

One of the responses exhibited by cyanobacteria when they are limited for an essential nutrient is the rapid degradation of their light-harvesting complex, the phycobilisome. Phycobilisome degradation is an ordered proteolytic process, visible by a color change of the cyanobacterial cell from blue-green to yellow-green (chlorosis). The small polypeptide NblA plays a key role in degradation of phycobilisomes in Synechococcus sp. PCC7942. Unlike Synechococcus, Synechocystis sp. PCC6803 has two nblA-homologous genes, nblA1 and nblA2, which are contiguous on the genome. Here we show that nblA1 and nblA2 are simultaneously expressed in Synechocystis 6803 upon nitrogen deprivation, and are both required for phycobilisome degradation.

Clinical trials in the gene therapy of arthritis.

Gene therapy clinical trials raise important safety issues that complicate their design and require extensive preclinical testing. Human protocols for the treatment of arthritis and most other orthopaedic and rheumatologic indications are complicated additionally by the perception that they are largely acquired, nonlethal conditions. Taking these considerations into account, the first such human study used the local, ex vivo delivery of a gene whose product, the interleukin-1 receptor antagonist, has an outstanding safety profile. This gene was delivered to the metacarpophalangeal joints of postmenopausal women 1 week before these joints were removed during total joint replacement surgery. In addition to providing an additional safety cushion, the surgical removal of the genetically modified joints made available large amounts of tissue to examine for evidence of successful gene transfer and gene expression. This Phase I safety study was approved at the local and federal levels, and its funding was contingent on the establishment of an external monitoring board. This trial now has been completed and a Phase II, efficacy study is being planned. A similar study has begun in Dusseldorf, Germany and results from the first two patients are similar to the results of the American patients. Permission has been given for two additional human trials, one in the United States and one in the Netherlands, in which a gene encoding herpes thymidine kinase will be transferred to the joints of patients with rheumatoid arthritis who then will be administered gancyclovir. This procedure aims to treat the disease by producing a genetic synovectomy. Additional development of human gene therapies for arthritis and other orthopaedic and rheumatic conditions will be aided by the successful completion of these studies.

[Bone density and bone metabolism regulated by vitamin D receptor allele polymorphism in a German study sample]. / Knochendichte und Knochenstoffwechsel unter dem Einfluss des Vitamin-D-Rezeptor-Allel-Polymorphismus in einem deutschen Untersuchungskollektiv.

BACKGROUND: It has been reported that the spinal bone density is associated with vitamin-D-receptor (VDR) gene polymorphisms. The results of recent studies have been contradictory concerning the predictive power for low bone mineral density (BMD). Regional population-specific influences have been found to affect the vitamin-D-endocrinologic-system, diminishing the influence of VDR polymorphism on BMD and on bone turnover. We have examined the association of bone density, fracture predictivity and biochemical markers of bone turnover with VDR polymorphism in a German population. METHODS: Blood and urine were collected from a heterogeneous subset of 164 caucasian subjects with ethnic German background. Polymerase chain reaction and subsequent digestion with Bsm I were used to examine variations of VDR genotypes. The morning following initial specimen collections, both urinary excretion rate of pyridinoline crosslinks (Pyr) and serum levels of bone alkaline phosphatase (BAP) were determined. For determination of the bone mineral density, the well-established method of dual X-ray absorptiometry was used. FINDINGS: The VDR BB-genotype was associated with low bone mineral density for age-matched subjects (90.1 +/- 15.5%) versus the bb-genotype (100.8 +/- 10.8%) at the lumbar spine and at the Ward's triangle (91.8 +/- 17.9% versus 101.9 +/- 12.1%). 34.2% of BB-genotype subjects, 14.2% of bB-genotype subjects and 12.6% of bb-genotype subjects had Z-score related bone mineral density < 85%. The fracture rate at typical osteoporotic fracture sites was 23.6% for the BB-, 10.8% for the bB- and 0.0% for the bb-genotype. The urinary excretion rate of free pyridinoline crosslinks was higher for the BB-genotype than for the bB- or the bb-, however the difference was not significant. No genotype specific variations were seen for bone alkaline phosphatase. INTERPRETATION: The authors conclude from this study that bone mineral density at the axial skeleton is associated with the vitamin-D-receptor allele polymorphism and that there is an influence on bone turnover and fracture rate in a German subset. CLINICAL RELEVANCE: The analysis of the VDR-genotype can be considered as a new piece in the puzzle of the diagnostic of osteoporosis for we get prognostic hints concerning the rate of fracture.

Current status of gene therapy for rheumatoid arthritis.

Despite the high prevalence of the disease, at present little effective pharmacological treatment of rheumatoid arthritis is available. Novel approaches utilising biological agents have resulted in the development of new antiarthritic and antiinflammatory agents, such as tumour necrosis factor-alpha (TNFalpha)-specific antibodies and interleukin-1 receptor antagonist (IL-1ra). Local gene therapy not only allows the pharmaceutical use of these biologicals, but also allows for continuous drug supply, which is necessary for chronic diseases like rheumatoid arthritis. We discuss the basics of rheumatoid arthritis therapy, candidate genes and possible gene transfer methods. A current clinical gene therapy trial is focusing on the IL-1 system using IL-1ra as a transgene. The transfer system, clinical protocol and preliminary results are described. After treatment of 11 patients we feel that gene therapy will offer potential as a new avenue to treat rheumatoid arthritis.

Spontaneous secretion of interleukin 1 receptor antagonist (IL-1ra) by cells isolated from herniated lumbar discal tissue after discectomy.

In the study presented, cells of a herniated lumbar disc were cultivated in vitro and analysed for interleukin 1beta (IL-1beta) and interleukin 1 receptor antagonist (IL-1Ra) production. The objective of this study was the detection of IL-1beta and IL-1Ra secreted by herniated lumbar discal cells after discectomy. The involvement of cytokines in the degeneration of intervertebral discs and in the pathophysiology of radiculopathy is established. Antagonizing proteins, e.g. IL-1Ra are thought to have considerable therapeutic potential. In the present study, a 51-year-old male with massive sequestrated lumbar disc herniation at L5/S1 was treated by microsurgical discectomy. Discal cells were isolated, cultures and culture supernatants immunochemically analysed for IL-1beta and IL-1Ra secretion. Spontaneous secretion of IL-1Ra was found. IL-1beta was not detected. Our findings might contradict recent studies on the role of IL-1beta and IL-1Ra. A possible therapeutic role of exogenous IL-1Ra in disc degeneration needs further research.

[Acupuncture together with cytokine depressing herbs in comparison to injection therapy with steroids in sciatic pain]. / Akupunktur kombiniert mit Naturstoffen in der Behandlung der Lumboischialgie. Einschätzung der Schmerzreduktion durch den Patienten und biologische Hintergründe.

INTRODUCTION: Because of their frequency and consequences sciatica remains a demanding clinical entity with significant social and economical impact. There is a high demand on therapeutic modalities, using folk medicine methods in the treatment of orthopaedic diseases. No data is available on the usefulness of methods like natural herbs in combination with acupuncture. Aim of our study was to present data on the effect of acupuncture and cytokine-inhibiting natural herbs in sciatic pain. We compared these results with nerve root infiltration by local anaesthetics and corticosteroids in our orthopaedic outpatient clinic. MATERIALS AND RESULTS: 278 patients with chronic pain for at least 3 months were observed. All three therapeutical modalities showed improvement of pain scores. Best results were gained with steroid injection ( n=26; 66% pain reduction), acupuncture in combination with herbs improved the pain in average of 62% ( n=230), whereas injection with local anaesthetic had a pain reduction of 48% ( n=22). Sole Mepivacain-HCl treatment had significant less pain reduction compared to the other treatment modalities. CONCLUSION: According to our results acupuncture in combination with herbs and steroid nerve blocks appear to be an effective and thus recommendable conservative therapy in cases of sciatic pain. Taking into account that patients increasingly prefer ethnomedical modalities of treatment our study gains importance for practising orthopaedists.

Transfer of genes to chondrocytic cells of the lumbar spine. Proposal for a treatment strategy of spinal disorders by local gene therapy.

STUDY DESIGN: In the current study, chondrocytic cells from bovine intervertebral end plates were cultivated in vitro and modified genetically. OBJECTIVE: The authors intended to perform isolation and cultivation of cells from bovine end plates of the spine. They also intended to show, in principle, the feasibility of introducing exogenous genes into chondrocytic cells from bovine intervertebral end plates by way of retroviral vectors. SUMMARY OF BACKGROUND DATA: The involvement of cytokines in the destruction of articular cartilage is established. It appears possible that similar mechanisms may play a role in intervertebral disc degeneration and other spinal disorders. Conventional medication and surgery of intervertebral disc degeneration addresses neither the pathophysiology nor the chronicity of the disease. Therapeutic proteins carry great potential as locally produced drugs after transfer of their cognate genes to the sites of interest. METHODS: Vertebral end plate tissue was obtained from bovine os coccygis. Chondrocytic cells were isolated and cultured in vitro. The bacterial beta-galactosidase (LacZ) gene and, alternatively, the complementary DNA (DNA copy of the mRNA) of the human interleukin-1 receptor antagonist were introduced into the isolated cells by retrovirus mediated gene transfer. beta-galactosidase activity was determined by staining, and interleukin-1 receptor antagonist protein was quantitated by enzyme-linked immunosorbent assay. RESULTS: Isolation and cultivation of chondrocytic end plate cells is possible. Native cells continue to grow in culture for more than 2 months. Transfer of the beta-galactosidase gene to cultured cells resulted in approximately 1% beta-galactosidase positive cells. Transfer of the interleukin-1 receptor antagonist complementary DNA resulted in the production of 24 ng/ml/10(6) cells interleukin-1 receptor antagonist protein in 48 hours. CONCLUSIONS: The introduction of exogenous therapeutic genes into cells from the intervertebral end plate opens the possibility for a local gene-based treatment of intervertebral disc degeneration. This approach avoids some of the shortcomings of conventional drug- and surgery-based treatments and has the potential to be specific, effective, and appropriate to the chronicity of the disease.

[In vitro transfer of genes in spinal tissue]. / In vitro Transfer von Genen in spinale Gewebe.

INTRODUCTION: It is well known that cytokines are involved in the induction of intervertebral disc and articular cartilage destruction. Therapeutic proteins are of great potential as locally produced drugs after transfer of their cognate genes to the sites of interest. METHODS: Chondrocytic cells from bovine os coccygis and discal chondrocytes from 6 wistar rats were isolated and cultured in vitro. The bacterial beta-galactosidase (LacZ) gene and the cDNA of the human interleukin-1 receptor antagonist (IL-1ra) were introduced into the cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1ra protein was determined by ELISA. RESULTS: Our study confirms that isolation and cultivation of bovine chondrocytic end plate cells and of rat discal cells in possible. Transfer of both LacZ and of the IL-1ra cDNA to cultured cells was successfull. DISCUSSION: The introduction of exogenous therapeutical genes into cells from the intervertebral disc and the end plate opens the possibility for a local gene therapy of IVD degeneration. This therapy has the potential to be specific, effective and appropriate to the chronicity of the disease.

[Genetic determination of bone density]. / Genetische Determinierung der Knochendichte.

Osteoporosis is a very common disease that concerns every forth postmenopausal white woman. The loss of bone density leads to fractures, mainly to fractures of the neck of the femur. A hereditary genesis of the idiopathic osteoporosis seems to be sure. The vitamin D receptor (VDR) has an important role for the mineralisation of the bones. His allelic variants can be used for a prediction of bone density. These alleles are correlated with endonuclease restriction areas for Bsm-1, Apa-1 and Taq-1. Among these Bsm-1 has the highest incidence for prediction. In this study the allelic variants of the VDR gene correlated with the bone density of the lumbar spine (p < 0.009).

Neurophysiologic changes in lumbar nerve root inflammation in the rat after treatment with cytokine inhibitors. Evidence for a role of interleukin-1.

STUDY DESIGN: The present study was designed to evaluate the effect of cytokine inhibitors in experimental allergic radiculitis. OBJECTIVE: Evaluation of the effect of cytokine inhibitors in experimental allergic radiculitis. SUMMARY OF BACKGROUND DATA: A number of cytokines are known to be involved in hyperalgesia and may play a role in radiculitis. Corticosteroids and other cytokine inhibitors antagonize their effects. METHODS: Experimental allergic radiculitis was induced in rats by injection of bovine myelin from the peripheral nervous system. The sham group subsequently received saline injections; the treatment groups received either prednisolone or interleukin-1 receptor antagonist. Treatment effect was assessed on the basis of motor performance and neurophysiologic parameters. RESULTS: Treatment ameliorated the symptoms of experimental allergic radiculitis. Prednisolone appeared to be somewhat more effective than interleukin-1 receptor antagonist. CONCLUSIONS: Because interleukin-1 receptor antagonist specifically blocks the effects of interleukin-1 at its receptors, the present results imply that interleukin-1 is a causal factor in the model of experimental radiculitis used. Its specificity and apparent lack of side effects make interleukin-1 receptor antagonist an attractive candidate treatment for the human disease.

Magnetic stimulation as a diagnostic tool in cervical nerve root compression and compression-induced neuropathy.

Magnetic stimulation of motor cortex and cervical spinal cord was used to assess motor impairment in patients with either cervical nerve root compression or myelopathy. Evoked potentials were recorded from biceps brachii and abductor pollicis brevis. Our interest centered on whether the paresis resulting from root compression is accompanied by an increased latency of magnetically evoked muscle potentials and whether latency is increased in cases of cervical root compression or myelopathy in which no paresis occurs. Latency increase does appear to be a moderately good indicator of disturbed nerve conduction. In particular, prolonged latencies in cases of "subclinical" paresis could be used as an important diagnostic tool for the early detection of motor deficits in cervical compression radiculopathy and myelopathy.

[Perspectives in gene therapy in rheumatic and degenerative diseases of the spine and joints]. / Perspektiven der Gentherapie bei rheumatischen und degenerativen Erkrankungen der Wirbelsäule und der Gelenke.

Rheumatoid degenerative conditions of the spine and other joints are most common in elderly patients. Data by the "National Center for Health Statistics" state that approximately 500 in 1000 persons above 65 are affected. Numbers for Germany should be in the same range. Although a lot of research has been dedicated to these conditions many of the actual causes remain unclear. There is consensus about the involvement of Interleukin-1 in the development of rheumatoid arthritis RA. Recent results point towards a more refined understanding and a gene therapy approach utilizing synovial cells. The synovium will in this strategy be the locus of drug (eg hormone) synthesis antagonizing the rheumatoid disease. We believe that this new approach will have a major impact on the treatment of RA.

[Receptor scintigraphy using 111In-pentetreotide in endocrine gastroenteropancreatic tumors]. / Rezeptorszintigraphie mit 111In-Pentetreotid bei endokrinen gastroenteropankreatischen Tumoren.

Receptor scintigraphy with 111In-pentetreotide is a complementary imaging technique with a sensitivity of 88% for the localization of the primary tumor and its metastases in patients presenting with the clinical and biochemical symptoms of an endocrine tumor of the gastrointestinal tract or the pancreas. As a whole-body scintigraphic technique it covers all body regions and is also able to reveal small tumors which can only be detected with difficulty or not at all by the usual imaging methods. In 104 patients with GEP tumors or after operative removal of such tumors, receptor scintigraphy proved to be superior to ultrasound and computed tomography in 34%, equal in 52% and inferior in 14% of the cases.

Receptor scintigraphy with 111In-pentetreotide for endocrine gastroenteropancreatic tumors.

Receptor scintigraphy with 111In-pentetreotide is a simple method with a sensitivity of 86% for the localization of the primary tumor and its metastases in patients presenting with the clinical and biochemical symptoms of an endocrine tumor of the gastrointestinal tract or the pancreas. As a whole-body scintigraphic technique it covers all body regions and is also able to reveal small tumors which either cannot be detected or can only be detected with difficulty by the usual imaging methods. In 85 patients with GEP tumors or after operative removal of such tumors, receptor scintigraphy proved to be superior to ultrasound and computed tomography in 34%, equal in 52%, and inferior in 14% of the cases.

Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins.

An excision assay system for En/Spm was developed in transgenic tobacco. The characteristics of excision and integration are similar to the natural system of Zea mays. In this transgenic model system two En/Spm encoded trans-acting functions, TNPA and TNPD, are required for excision. A biochemical model for transposition is proposed that might also be applicable to other transposable elements.