Does sagittal position of the CTDR-related centre of rotation influence functional outcome? Prospective 2-year follow-up analysis.

PURPOSE: Recent studies describe significant rates of heterotopic ossification (HO) after cervical total disc replacement (CTDR). Little is known about the reasons, and one aspect that requires further in vivo investigation is the biomechanical alteration after CTDR and the role of the implant-related centre of rotation (CORi) in particular. The role of the sagittal position of the CORi on functional outcome in two versions of a semi-constrained disc prosthesis with sagittally different CORi is the topic of this study. METHODS: Patients were candidates for single-level CTDR between C3 and C7 who suffered from CDDD and received a standard or flat version of activ C™ (Aesculap AG, Tuttlingen). Clinical and radiographic assessments were determined preoperatively, intraoperatively, at discharge and again at 6 weeks, 6 months, 1 and 2 years. Radiographic examinations were performed independently using specialized quantitative motion analysis software. RESULTS: Clinical outcome improved significantly regarding NDI as well as VAS on neck and arm pain with no differences in mean improvement by study group. Segmental angle measures show a significantly better lordotic alignment for both groups after surgery, but the degree of correction achieved is higher in the flat group. Correlation analysis proves that the more anterior the CORi is positioned, the higher the lordotic correction is achieved (Pearson rho -0.385). Segmental ROM decreased in the standard group but was maintained for flat implants. At present, our data do not demonstrate a correlation between CORi and ROM at 2 years. Two years after surgery, severe HO grade III-IV was present in 31.6 % standard and 13.1 % flat cases with significant differences. Grouping according to HO severity showed comparable sagittal positions of CORi for flat implants but a more posterior position in the severe HO group for standard implants. CONCLUSIONS: Our results confirm the influence of CORi location on segmental alignment, kinematics and HO for a semi-constrained CTDR, but it also indicates a multifactorial process.

Reactivation of hepatitis E infection in a patient with acute lymphoblastic leukaemia after allogeneic stem cell transplantation.

Hepatitis E virus (HEV) is the major cause of several outbreaks of waterborne hepatitis in tropical and subtropical countries and of sporadic cases of viral hepatitis in endemic and industrialised countries. Generally, HEV causes an acute self-limiting hepatitis. The clinical course is characterised by transient viraemia and transaminasaemia followed by a full hepatic recovery. Recent studies describe prolonged and chronic HEV infections in some immunosuppressed patients after solid organ transplantation. Here, an indigenous acute limited hepatitis E in a patient with Philadelphia chromosome-positive acute lymphoblastic leukaemia prior to allogeneic stem cell transplantation is reported. Fourteen weeks after stem cell transplantation, reappearance of HEV viraemia was observed, with increasing viral load and modestly elevated serum transaminases. Sequence analysis of the viral RNAs revealed a reactivation of endogenous HEV genotype 3, indicating viral persistence after recovery from acute hepatitis E.

Indigenous hepatitis E virus infection of a plasma donor in Germany.

BACKGROUND: Although Europe is supposed to be non-endemic for hepatitis E virus (HEV), locally acquired human cases are registered, and a relatively high prevalence for anti-HEV was found in blood donors in some European countries. Transfusion-transmitted infections by contaminated blood products were reported in Japan and sporadically in Europe. MATERIALS AND METHODS: Several samples from a plasma donor were screened with a highly sensitive quantitative HEV real-time polymerase chain reaction and the full-length genome was generated. Serology was performed with two different commercially available ELISA kits. RESULTS: The full-length genome sequence of human HEV was identified using samples from a plasma donor with acute self-limiting hepatitis. Plasma donated 2 weeks before onset of elevated liver enzyme levels was already positive for HEV RNA (10(4) copies/ml). High viraemia (10(6) copies/ml) correlated with the detection of anti-HEV IgM in the first blood sample with increased alanine transaminase levels. Phylogenetic analyses grouped the isolate within genotype 3, subtype 3f. CONCLUSION: The sequence analyses and the epidemiological data revealed that the plasma donor was most probably infected with a swine HEV. This case supports the ongoing discussion of an obligatory HEV nucleic acid testing of blood products for special recipient risk groups.

Short communication: bovine kappa-casein variants result in different angiotensin I converting enzyme (ACE) inhibitory peptides.

Proteins in bovine milk are a common source of bioactive peptides. The peptides are released by the digestion of caseins and whey proteins. Peptides derived from the different genetic variants A, B, C, E, F1, F2, G1, G2, H, I, and J of bovine kappa-casein (CSN3) were investigated for their inhibitory activities against angiotensin I converting enzyme (ACE). Amino acid sequences of the CSN3 variants were analyzed in silico to detect potential ACE inhibitory peptides. Besides known biologically active peptides, exclusive peptides were identified in some CSN3 variants and their biological activity was determined: within CSN3*B and CSN3*C, the ACE inhibitory peptide ASP (IC50 = 242.3; the IC50 value is equivalent to the micromolar concentration of peptide mediating a 50% inhibition of ACE activity) and within CSN3*C the peptide AHHP (IC50 = 847.6) was detected. Furthermore, the peptides VSP (IC50 = 21.8) and ACHP (IC50 = 360.7) were identified in CSN3*F1 and CSN3*G2, respectively.

Cell transplantation in lumbar spine disc degeneration disease.

Low back pain is an extremely common symptom, affecting nearly three-quarters of the population sometime in their life. Given that disc herniation is thought to be an extension of progressive disc degeneration that attends the normal aging process, seeking an effective therapy that staves off disc degeneration has been considered a logical attempt to reduce back pain. The most apparent cellular and biochemical changes attributable to degeneration include a decrease in cell density in the disc that is accompanied by a reduction in synthesis of cartilage-specific extracellular matrix components. With this in mind, one therapeutic strategy would be to replace, regenerate, or augment the intervertebral disc cell population, with a goal of correcting matrix insufficiencies and restoring normal segment biomechanics. Biological restoration through the use of autologous disc chondrocyte transplantation offers a potential to achieve functional integration of disc metabolism and mechanics. We designed an animal study using the dog as our model to investigate this hypothesis by transplantation of autologous disc-derived chondrocytes into degenerated intervertebral discs. As a result we demonstrated that disc cells remained viable after transplantation; transplanted disc cells produced an extracellular matrix that contained components similar to normal intervertebral disc tissue; a statistically significant correlation between transplanting cells and retention of disc height could displayed. Following these results the Euro Disc Randomized Trial was initiated to embrace a representative patient group with persistent symptoms that had not responded to conservative treatment where an indication for surgical treatment was given. In the interim analyses we evaluated that patients who received autologous disc cell transplantation had greater pain reduction at 2 years compared with patients who did not receive cells following their discectomy surgery and discs in patients that received cells demonstrated a significant difference as a group in the fluid content of their treated disc when compared to control. Autologous disc-derived cell transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration. Adipose tissue provides an alternative source of regenerative cells with little donor site morbidity. These regenerative cells are able to differentiate into a nucleus pulposus-like phenotype when exposed to environmental factors similar to disc, and offer the inherent advantage of availability without the need for transporting, culturing, and expanding the cells. In an effort to develop a clinical option for cell placement and assess the response of the cells to the post-surgical milieu, adipose-derived cells were collected, concentrated, and transplanted under fluoroscopic guidance directly into a surgically damaged disc using our dog model. This study provides evidence that cells harvested from adipose tissue might offer a reliable source of regenerative potential capable of bio-restitution.

Total dura substitute in the spine: double layer dural substitute made from polylactide layer and bovine pericardium.

When there is significant loss of spinal dura mater, dural substitution with synthetic or allogenic materials is essential. In the case of laminectomy, mechanical protection and reformation of the dorsal spinal canal may be useful. This is a report on a patient with total dura loss through tumour atrophy of the dura and laminae. In order to reconstruct the dorsal face of the spinal canal a polylactide sheet was cut and shaped to fit the physiological contour. A bovine dura substitute was firmly attached and sutured to the inner surface of the polylactide shield. The implant was wedged in between the pedicles and the facet joints and resulted in a water-tight dura substitute maintaining the shape of the spinal canal and protecting it against mechanical forces and intradural scar formation.

Calcium Microcrystal Formation in Recurrent Herniation Patients After Autologous Disc Cell Transplantation.

Autologous disc cell transplantation (ADCT) is a cell-based therapy aiming to initiate regeneration of intervertebral disc (IVD) tissue, but little is known about potential risks. This study aims to investigate the presence of structural phenomena accompanying the transformation process after ADCT treatment in IVD disease. Structural phenomena of ADCT-treated patients (Group 1, n = 10) with recurrent disc herniation were compared to conventionally-treated patients with recurrent herniation (Group 2, n = 10) and patients with a first-time herniation (Group 3, n = 10). For ethical reasons, a control group of ADCT patients who did not have a recurrent disc herniation was not possible. Tissue samples were obtained via micro-sequestrectomy after disc herniation and analyzed by micro-computed tomography, scanning electron microscopy, energy dispersive spectroscopy, and histology in terms of calcification zones, tissue structure, cell density, cell morphology, and elemental composition. The major differentiator between sample groups was calcium microcrystal formation in all ADCT samples, not found in any of the control group samples, which may indicate disc degradation. The incorporation of mineral particles provided clear contrast between the different materials and chemical analysis of a single particle indicated the presence of magnesium-containing calcium phosphate. As IVD calcification is a primary indicator of disc degeneration, further investigation of ADCT and detailed investigations assessing each patient's Pfirrmann degeneration grade following herniation is warranted. Structural phenomena unique to ADCT herniation prompt further investigation of the therapy's mechanisms and its effect on IVD tissue. However, the impossibility of a perfect control group limits the generalizable interpretation of the results.

Parvovirus B19 infection associated with unilateral cervical lymphadenopathy, apoptotic sinus histiocytosis, and prolonged fatigue.

This report describes the case of a 16 year old girl with a history of high fever, prolonged fatigue, and cervical lymphadenopathy of the right side. In addition, the patient showed neutropenia, thrombopenia, and pronounced reticulopenia. Cervical ultrasound showed unilateral hypoechoic lymph nodes up to 23 mm in diameter suspicious for malignant lymphoma. Histology of a cervical lymph node specimen revealed massive nodular histiocytic proliferation and prominent apoptosis without necrosis. Parvovirus B19 was detected by polymerase chain reaction and immunohistochemistry in the lymph node. In summary, this case is an unusual presentation of parvovirus B19 infection. The virus was identified as the potential causative agent of unilateral cervical lymphoma and apoptotic sinus histocytosis, thus broadening the clinicopathological spectrum of parvovirus B19 induced diseases.

Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein.

The in vivo formation of an opioid peptide (exorphin) derived from beta-casein has been proved for the first time. It was isolated from duodenal chyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a beta-casein fragment by end-group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named beta-casomorphin-11, displayed substantial opioid activity in an opiate receptor-binding assay.

Stimulation of human peripheral blood lymphocytes by bioactive peptides derived from bovine milk proteins.

The in vitro modulation of the proliferation of human peripheral blood lymphocytes by different synthetic peptides derived from milk proteins was investigated. Therefore, proliferation changes were followed up after incorporation of BrdU into the DNA, and the influence on protein biosynthesis was measured using the [3H]leucine incorporation test. Tyr-Gly and Tyr-Gly-Gly significantly enhanced (maximal 90 and 35%, respectively) the proliferation of PBL. For beta-casomorphin-7 and beta-casomorphin-10,lymphocyte proliferation was suppressed at lower concentrations, but stimulated at higher concentrations (> or = 10(-7) mol/l). Protein synthesis was stimulated (maxima at 25%) only with Tyr-Gly and Tyr-Gly-Gly. The findings point to a need for further studies on the possible function of peptides derived from milk proteins as orally bioavailable immunopotentiatory compounds.

Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine beta-lactoglobulin.

The angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine beta-lactoglobulin (beta-lg) was investigated. Intact beta-lg essentially did not inhibit ACE while the tryptic digest gave an 84.3% inhibition of ACE. Peptide material eluting between 20 and 25% acetonitrile during C18 solid-phase extraction of the beta-lg tryptic digest inhibited ACE by 93.6%. This solid-phase extraction fraction was shown by mass spectroscopy to contain beta-lg f(142-148). This peptide had an ACE IC50 value of 42.6 micromol/l. The peptide was resistant to further digestion with pepsin and was hydrolysed to a very low extent with chymotrypsin. The contribution of specific amino acid residues within the peptide to ACE inhibitory activity and the potential application of this peptide as a nutraceutical is discussed.

Apoptosis induced by modified ribonucleosides in human cell culture systems.

The in vitro modulation of apoptosis and cell proliferation by modified in comparison with non-modified ribonucleosides was investigated for the first time using peripheral blood lymphocytes, HL-60 cells and Caco-2 cells as human cell culture models. Modulating effects of several ribonucleosides were found in the range of 10(-7)-10(-3) mol/l. The following ribonucleosides induced significant apoptosis of HL-60 cells: adenosine, N6-dimethyladenosine, N6-(2-isopentenyl)-adenosine, N2-dimethylguanosine. A significant apoptotic effect on PBL was found with N6-dimethyladenosine and N6-(2-isopentenyl)-adenosine. N6-Dimethyladenosine, N6-(2-isopentenyl)-adenosine and guanosine had a pronounced inhibitory effect on Caco-2 cell apoptosis. Regarding the known function of ribonucleosides as pathobiochemical marker molecules for cancer, the possibility of a selective apoptotic effect against malignant cells is discussed.

Post-translational phosphorylation affects the IgE binding capacity of caseins.

IgE response specific to those molecular regions of casein that contain a major phosphorylation site was analyzed using native and modified caseins and derived peptides. This study included (i) the naturally occurring common variants A1 and A from beta- and alphas2-caseins, respectively, which were purified in the native form and then dephosphorylated, (ii) a purified rare variant D of alphas2-casein which lacks one major phosphorylation site, and (iii) the native and dephosphorylated tryptic fragment f(1-25) from beta-casein. Direct and indirect ELISA using sera from patients allergic to milk showed that the IgE response to caseins is affected by modifying or eliminating the major phosphorylation site.

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles.

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.

Biofunctional peptides from milk proteins: mineral binding and cytomodulatory effects.

The protein fraction of milk contains many valuable components and biologically active substances. Moreover, milk proteins are precursors of many different biologically active peptides which are inactive within the sequence of the precursor protein but can be released by enzymatic proteolysis. Many milk protein-derived peptides, such as caseinophosphopeptides, reveal multi-functional bioactivities. Caseinophosphopeptides can form soluble organophosphate salts and may function as carriers for different minerals, especially calcium. Furthermore, they have been shown to exert cytomodulatory effects. Cytomodulatory peptides inhibit cancer cell growth or they stimulate the activity of immunocompetent cells and neonatal intestinal cells, respectively. Several bioactive peptides derived from milk proteins are potential modulators of various regulatory processes in the body and thus may exert beneficial physiological effects. Caseinophosphopeptides are already produced on an industrial-scale and as a consequence these peptides have been considered for application as ingredients in both 'functional foods' and pharmaceutical preparations. Although the physiological significance as exogenous regulatory substances is not yet fully understood, both mineral binding and cytomodulatory peptides derived from bovine milk proteins are claimed to be health enhancing components that can be used to reduce the risk of disease or to enhance a certain physiological function.

Mapping of B-cell epitopes in the nucleocapsid protein of Puumala hantavirus.

Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/Hällnäs, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/Hällnäs and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined.

Short report: simultaneous occurrence of Dobrava, Puumala, and Tula Hantaviruses in Slovakia.

The prevalence of antibody to hantaviruses in Slovakia (serum panel n = 2,133) was lower in the western part (0.54%) and higher in the eastern part (1.91%) of the country and was found to be significantly enhanced in a group of forest workers from eastern Slovakia (5.88%). One-third of the IgM-negative convalescent phase sera from patients with hemorrhagic fever with renal syndrome exhibited antibodies reacting predominantly with Puumala virus antigen, while two-thirds had antibodies directed mainly against Hantaan virus antigen. Fine analysis of two Hantaan virus-reactive sera by a focus reduction neutralization test showed that Dobrava hantavirus was the source of these human infections. Initial results of rodent screening indicated the circulation of Dobrava virus in populations of striped field mice (Apodemus agrarius) in eastern Slovakia.

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection.

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/Hällnäs into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.