Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.
BackgroundNeuroborreliosis represents a relevant infectious disease and can cause a variety of neurological manifestations. Different stages and syndromes are described and atypical symptoms can result in diagnostic delay or misdiagnosis. The aim of this retrospective study was to define the pivotal neurological deficits in patients with neuroborreliosis that were the reason for admission in a hospital.MethodsWe retrospectively evaluated data of patients with neuroborreliosis. Only patients who fulfilled the diagnostic criteria of an intrathecal antibody production against Borrelia burgdorferi were included in the study.ResultsSixty-eight patients were identified with neuroborreliosis. Cranial nerve palsy was the most frequent deficit (50%) which caused admission to a hospital followed by painful radiculitis (25%), encephalitis (12%), myelitis (7%), and meningitis/headache (6%). In patients with a combination of deficits, back pain was the first symptom, followed by headache, and finally by cranial nerve palsy. Indeed, signs of meningitis were often found in patients with neuroborreliosis, but usually did not cause admission to a hospital. Unusual cases included patients with sudden onset paresis that were initially misdiagnosed as stroke and one patient with acute delirium. Cerebrospinal fluid (CSF) analysis revealed typical changes including elevated CSF cell count in all but one patient, a blood-CSF barrier dysfunction (87%), CSF oligoclonal bands (90%), and quantitative intrathecal synthesis of immunoglobulins (IgM in 74%, IgG in 47%, and IgA in 32% patients). Importantly, 6% of patients did not show Borrelia specific antibodies in the blood.ConclusionIn conclusion, the majority of patients presented with typical neurological deficits. However, unusual cases such as acute delirium indicate that neuroborreliosis has to be considered in a wide spectrum of neurological diseases. CSF analysis is essential for a reliable diagnosis of neuroborreliosis.
BackgroundVaricella zoster virus (VZV) reactivation is a common infectious disease in neurology and VZV the second most frequent virus detected in encephalitis. This study investigated characteristics of clinical and laboratory features in patients with VZV infection.MethodsTwo hundred eighty two patients with VZV reactivation that were hospitalized in the department of neurology in the time from 2005 to 2013 were retrospectively evaluated. Results from cerebrospinal fluid (CSF) analysis were available from 85 patients.ResultsTrigeminal rash was the most common clinical manifestation, followed by segmental rash, CNS infection, facial nerve palsy, postherpetic neuralgia, and radiculitis. MRI of the brain performed in 25/33 patients with encephalitis/meningitis did not show any signs of infection in the brain parenchyma. Only one patient showed contrast enhancement in the hypoglossal nerve. General signs of infection such as fever or elevated CRP values were found in only half of the patients. Furthermore, rash was absent in a quarter of patients with CNS infection and facial nerve palsy, and thus, infection could only be proven by CSF analysis. Although slight inflammatory CSF changes occurred in few patients with isolated rash, the frequency was clearly higher in patients with CNS infection and facial nerve palsy.ConclusionMonosegmental herpes zoster is often uncomplicated and a diagnostic lumbar puncture is not essential. In contrast, CSF analysis is an essential diagnostic tool in patients with skin lesions and cranial nerve or CNS affection. In patients with neuro-psychiatric symptoms and inflammatory CSF changes analysis for VZV should be performed even in the absence of skin lesions.
Background: Sjögren's syndrome is a chronic autoimmune-mediated disease that can cause a variety of neurological manifestations. Methods: This study investigated characteristics of clinical and cerebrospinal fluid (CSF) features in patients with neurological diseases associated with Sjögren's syndrome. Eighty-two patients were examined separately according to the presence of Sjögren's syndrome alone or in combination with other autoimmune diseases. Results: In the 47 patients with primary Sjögren's syndrome, peripheral neuropathy (57%) was found most frequently, followed by the involvement of the central nervous system (CNS; 17%), cranial neuropathy (15%), and myalgia (11%). These patients did not display consistent signs of inflammation in the CSF. Slight pleocytosis of 8-107 cells/µL was found in patients with peripheral neuropathy (9%), cranial neuropathy (20%), and CNS involvement (25%). Oligoclonal bands indicating intrathecal IgG synthesis occurred in 26% of patients with peripheral neuropathy, 20% of patients with cranial neuropathy, and 25% of patients with CNS involvement. Conclusions: In patients with Sjögren's syndrome and neurological manifestations, inflammatory CSF changes were rarely found and did not show a characteristic pattern irrespective of peripheral or central genesis of neurological deficits. Analysis of the CSF presents therefore an important diagnostic procedure to exclude other autoimmune and infectious diseases.
Cryptococcal meningoencephalitis represents a serious infection of the central nervous system, where reliable prognostic factors during the disease course are needed. Twenty-one patients diagnosed with cryptococcal meningoencephalitis in a German university hospital from 1999 to 2013 were analysed retrospectively. CSF parameters were analysed prior to therapy and during antifungal treatment and were compared between patients who survived or deceased. Fifteen patients clinically improved after antifungal therapy, while six patients died. No differences were observed between the outcome groups for the CSF parameters cell count, lactate, total protein, and CSF-serum albumin quotients (QAlb). Follow-up examinations of serum cryptococcal antigen titer and CSF cell count have shown that these parameters cannot be used to monitor the efficacy of antifungal therapy as well. In contrast, the course of QAlb during therapy was indicative for the outcome as a possible prognostic marker. In patients with clinical improvement QAlb values were falling under therapy, while rising QAlb values were found in patients with fatal outcome indicating a continuing dysfunction of the blood-CSF barrier. In conclusion, our results indicate that, among the various CSF parameters, the course of QAlb presents a promising marker that might be used to monitor the efficacy of antifungal therapy.
Dimethylfumarate (DMF) has been approved the for treatment of relapsing-remitting multiple sclerosis. The mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood, notably for brain resident cells. Therefore we investigated potential direct effects of DMF and MMF on microglia and indirect effects on oligodendrocytes. Primary rat microglia were differentiated into M1-like, M2-like and M0 phenotypes and treated in vitro with DMF or MMF. The gene expression of pro-inflammatory and anti-inflammatory factors such as growth factors (IGF-1), interleukins (IL-10, IL-1β), chemokines (CCl3, CXCL-10) as well as cytokines (TGF-1β, TNFα), iNOS, and the mannose receptor (MRC1) was examined by determining their transcription level with qPCR, and on the protein level by ELISA and FACS analysis. Furthermore, microglia function was determined by phagocytosis assays and indirect effects on oligodendroglial proliferation and differentiation. DMF treatment of M0 and M1-like polarized microglia demonstrated an upregulation of gene expression for IGF-1 and MRC1, but not on the protein level. While the phagocytic activity remained unchanged, DMF and MMF treated microglia supernatants led to an enhanced proliferation of oligodendrocyte precursor cells (OPC). These results suggest that DMF has anti-inflammatory effects on microglia which may result in enhanced proliferation of OPC.
BackgroundParaneoplastic syndromes are serious immune caused diseases of the peripheral and/or central nervous system associated with malignant neoplasm. Symptoms develop when antibodies against antigens expressed by tumor cells cross-react with neuronal proteins. Antineuronal antibodies are usually examined in patient’s sera while examination of the cerebrospinal fluid (CSF) often fails. Furthermore, the few previous reports describing CSF data summarized different antineuronal antibodies and/or regarded patients with different neurological symptoms as one group.MethodsWe retrospectively evaluated data of 18 patients with paraneoplastic syndromes due to anti-Hu antibodies. The study aimed to differentiate patients with peripheral neuropathy and encephalitis by cerebrospinal fluid (CSF) parameters including anti-Hu antibody titers.ResultsOur results confirm previous observations that serum titers of anti-Hu antibodies and standard CSF values do not differ between patients with neuropathy and encephalitis. However, analysis of CSF anti-Hu titers and calculating the intrathecal synthesis helped to discriminate between both groups.ConclusionIn conclusion, our results indicate that patients even with one defined antineuronal antibody need to be regarded separately depending on the involved location of the nervous system. We recommend incorporation of anti-Hu analyses in the CSF and calculating the intrathecal synthesis in patients with anti-Hu syndrome.
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