Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy

Dendritic cells (DC) are increasingly explored as cellular vaccines for tumor immunotherapy. In most reported DC-based cancer vaccine trials, DC have been pulsed with soluble tumor antigen-derived peptide ligands of MHC molecules. Considering that the half-life of peptide/MHC complexes on the cell surface is relatively short and that soluble exogenous protein antigens cannot be efficiently processed via the MHC class I-processing pathway, the current vaccination procedure is not optimal for the induction of strong T cell responses aiming at tumor rejection. Recently, we have shown that antigen presentation can be prolonged when synthetic peptides were encapsulated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS) for uptake by DC. In the present study, we investigated the phenotypic and functional consequences of MS uptake by human monocyte-derived dendritic cells (MoDC) in vitro. We found that immature MoDC that were prepared in serum free media suitable for clinical application were able to phagocytose high numbers of MS, while matured MoDC showed a reduced capacity for phagocytosis of MS. The ingestion of MS did not change the cell surface expression of CD80, CD83, CD86 and HLA-DR of immature and mature DC, suggesting that MS uptake did not induce DC maturation but that maturation by cytokines or LPS was unaltered in the presence of MS. Furthermore, MS-loaded mature MoDC expressed normal levels of the chemokine receptor CCR7 and migrated as efficiently towards CCL19 or CCL21 as unloaded MoDC. DC viability and the secretion of TNF-alpha and IL-12 was not significantly changed by MS loading. Taken together, our data indicate that PLGA-MS loading has no negative effects on the pivotal properties of MoDC in vitro. It should therefore be feasible to further develop this antigen loading strategy for clinical use in immunotherapy against viral infections and tumors.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

Brucella lumazine synthase elicits a mixed Th1-Th2 immune response and reduces infection in mice challenged with Brucella abortus 544 independently of the adjuvant formulation used

The immunogenicity and protective efficacy of recombinant lumazine synthase from Brucella spp. (rBLS) administered with different adjuvants was evaluated in mice. Mice were immunized with rBLS in the absence or the presence of aluminum hydroxide gel (BLS-Al), monophosphoryl lipid A (BLS-MPA), or incomplete Freund's adjuvant (BLS-IFA). rBLS per se induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. All the adjuvants increased this response; the BLS-IFA formulation was the most effective at inducing BLS-specific IgG antibodies. In addition, after in vitro stimulation with rBLS, spleen cells from BLS-IFA-, BLS-Al-, or BLS-MPA-immunized mice proliferated and produced interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-10, and IL-4, suggesting the induction of a mixed Th1-Th2 response. Immunization with rBLS protected mice against challenge with B. abortus 544. The levels of protection in the spleen were similar for all adjuvants, but only BLS-Al and BLS-IFA were effective in the liver. Our results indicate that BLS might be a useful candidate for the development of subunit vaccines against brucellosis, since it elicits antigen-specific cellular responses, with production of IFN-gamma and protection, independently of the adjuvant formulation used.     

Prevalence of viral infection markers by polymerase chain reaction amplification and interferon-alpha measurements among patients undergoing lumbar puncture in an emergency department

Aseptic meningitis is a frequent diagnosis in emergency departments. Nevertheless, viral investigations are not carried out currently and the viral etiology in adult population has not been studied extensively. We conducted a prospective study including all consecutive patients undergoing lumbar puncture during a 15 months period in an adult emergency department. Bloody and purulent cerebrospinal fluid (CSF) were excluded. The main tests undertaken were: CSF genomic amplification by the polymerase chain reaction (PCR) for neurotropic viruses and serum and CSF interferon-alpha (IFN-alpha) measurements. Among 194 patients included, 45 had and 149 did not have aseptic meningitis. Of 45 patients with aseptic meningitis, 10 had alternative non-virological final diagnosis, and 35/45 were presumed to have neurological disorders of viral origin. Patients (27/35) completed virological analysis: 21/27 (78%) had either positive viral PCR (enterovirus: 8 patients, Varicella zoster virus (VZV): 5, Epstein-Barr virus (EBV): 2, herpes simplex virus (HSV): 1, human herpes virus 6: 1) or only raised serum or CSF IFN-alpha (4 patients). Overall, 59% of patients with a positive viral PCR had either CSF or serum raised IFN-alpha. Twentyone patients without meningitis had either positive viral PCR (enterovirus: 3 patients) or only high serum IFN-alpha level (18 patients). In the setting of aseptic meningitis diagnosed in an adult emergency department, viruses are the most common agents encountered, with enterovirus and VZV as the two main etiological agents. Current CSF viral genome amplification and IFN-alpha measurement are informative and could be useful to confirm the viral origin of various neurological disorders, although the sensitivity and specificity of IFN-alpha measurement for the diagnosis of viral infection need further confirmation.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Isolation of Bartonella washoensis from a dog with mitral valve endocarditis

We report the first documented case of Bartonella washoensis bacteremia in a dog with mitral valve endocarditis. B. washoensis was isolated in 1995 from a human patient with cardiac disease. The main reservoir species appears to be ground squirrels (Spermophilus beecheyi) in the western United States. Based on echocardiographic findings, a diagnosis of infective vegetative valvular mitral endocarditis was made in a spayed 12-year-old female Doberman pinscher. A year prior to presentation, the referring veterinarian had detected a heart murmur, which led to progressive dyspnea and a diagnosis of congestive heart failure the week before examination. One month after initial presentation, symptoms worsened. An emergency therapy for congestive heart failure was unsuccessfully implemented, and necropsy evaluation of the dog was not permitted. Indirect immunofluorescence tests showed that the dog was strongly seropositive (titer of 1:4,096) for several Bartonella antigens (B. vinsonii subsp. berkhoffii, B. clarridgeiae, and B. henselae), highly suggestive of Bartonella endocarditis. Standard aerobic and aerobic-anaerobic cultures were negative. However, a specific blood culture for Bartonella isolation grew a fastidious, gram-negative organism 7 days after being plated. Phenotypic and genotypic characterizations of the isolate, including partial sequencing of the citrate synthase (gltA), groEL, and 16S rRNA genes, indicated that this organism was identical to B. washoensis. The dog was seronegative for all tick-borne pathogens tested (Anaplasma phagocytophilum, Ehrlichia canis, and Rickettsia rickettsii), but the sample was highly positive for B. washoensis (titer of 1:8,192) and, according to indirect immunofluorescent-antibody assay, weakly positive for phase II Coxiella burnetii infection.     

Beeinflussung der Atemsteigerung bei Muskelarbeit durch partiellen neuromuskulären Block (Tubocurarin)

Zusammenfassung In 17 Versuchen erhielten 7 Versuchspersonen 0,09–0,16 (Mittel 0,13) mg Tubocurarin/kg i.v. Vor und nach der Injektion wurde die gleiche Muskelarbeit geleistet (Beinarbeit; O₂-Aufnahme etwa das dreifache des Ruheumsatzes). Gemessen wurden Atemzeitvolumen, alveolarer O₂- und CO₂-Druck, Blutdruck und Pulsfrequenz. Die Curaredosen führten zu einer Erschwerung der Arbeit, beeinträchtigten jedoch nicht die Atmung.Bei gleicher Muskelarbeit ventilierte die Mehrzahl der Versuchspersonen, vor allem im Beginn der Arbeit, unter Curare mehr als bei der Kontrollarbeit ohne Curare. Am deutlichsten war dies bei denjenigen Versuchspersonen ausgeprägt, die schon ohne Curare im Beginn der Arbeit eine plötzliche Steigerung der Ventilation zeigten.Es wird angenommen, daß unter Curare die gleiche Muskelarbeit mit einer verstärkten motorischen Innervation geleistet wird. Die dabei beobachtete Ventilationssteigerung wird als weiteres Argument für einen Einfluß der motorischen Innervation auf die Atemzentren angesehen.Mit 3 TextabbildungenHerrn Professor Dr. Hans Winterstein zum 80. Geburtstag gewidmet.