The EORTC Melanoma Group exchange program: evaluation of a multicenter monoclonal antibody study

In the framework of the European Organisation for Research and Treatment of Cancer (EORTC), the Immunology and Pathology Subgroups of the Malignant Melanoma Cooperative Group undertook a large multicenter monoclonal antibody (MAb) study. Fourteen laboratories from 7 European countries tested a panel of 23 MAbs for immunohistological staining reactivity for malignant and non-malignant lesions involving the melanocytic lineage. A standardized immunoperoxidase procedure was used and the results were evaluated using a standard protocol and data evaluation form developed in collaboration with the EORTC Data Center. According to this analysis, the antibodies in the panel could be classified into 3 main groups. The first group of MAbs includes those antibodies which stained the majority (greater than 80%) of all primary tumors, irrespective of their Breslow thickness and the majority of metastatic lesions. In addition, these MAbs stained a high percentage of cells within a given lesion. Several antibodies of Group I were likewise reactive with the majority of naevoblasts and with normal melanocytes. The second group of MAbs included antibodies reacting only with a limited number of primary melanomas and metastatic lesions. Antibodies of Group II reacted only weakly, if at all, with normal melanocytes or naevocytes. The percentage of cells within a malignant lesion stained by these MAbs was always rather low. The MAb group III detected surface structures whose expression appeared to be related to tumor progression; they did not react or reacted only weakly with naevi, and they all reacted with a small number of early primary melanomas (less than 0.75 mm). The number of lesions stained increased with increasing Breslow thickness. Our study suggests that the application of a panel of well defined MAbs might be of diagnostic and prognostic value in evaluating malignant melanoma.     

Superficial siderosis of the central nervous system

The course of a patient suffering from superficial siderosis of the central nervous system for 37 years is presented and diagnostic and therapeutic approaches are evaluated. The syndrome is clinically defined by slowly progressing deafness, cerebellar ataxia, myelopathy and neuropsychological deficits in combination with recurrent xanthochromia of the cerebrospinal fluid with siderophages. The diagnosis may be confirmed by computed tomography, which shows degeneration of the cerebellar vermis, and by magnetic resonance imaging, demonstrating iron deposits on the surface of brain, brain stem and spinal cord. Therapy should seek to identify and remove the source of bleeding, since pharmacotherapy with iron-depleting drugs is of limited effectiveness.     

Does a cycling program combined with education and followed by coaching promote physical activity in subacute stroke patients? A randomized controlled trial

Background: To investigate the effects of a three month active cycling program followed by coaching on physical activity in subacute stroke patients.

Methods: Patients (n?=?59; mean age =65.4?±?10.3) aged ≤80?years with first stroke and able to cycle at 50 revolutions/minute enrolled 3–10?weeks post stroke. Patients were randomly allocated to three month active cycling group (n?=?33) or to a control group (n?=?26), 3?x?30?minutes training/week. Afterwards, the active cycling group was randomized into a coaching (n?=?15) versus non-coaching group (n?=?16) for nine months. Physical activity was measured by objective and self-reported measures, which were taken before/after the active cycling program and during six and 12?months, except the Baecke-questionnaire, which was used at baseline and 12?months.

Results: A significant difference was found in Baecke/sport (95% confidence interval: 0.06, 2.24; p?=?0.039) between the active cycling group and the control group, in patients with severe motor function deficits at baseline. Patients in the control group performed significant less sports at 12?months (mean Baecke/sport_baseline =3.07?±?1.21, mean Baecke/sport_12months?=?1.43?±?0.98; p?=?0.01). Furthermore, all groups showed significant changes over time in all measures at three months (except: Physical Activity Scale for Individuals with Physical Disabilities, diary/Mets*minutes-moderate) and 12?month and additionally in a subgroup with severe motor function deficits (except diary Mets*minutes-sedentary).

Conclusion: When active cycling combined with education is used in subacute patients with severe motor function deficits, more sport participation might be observed after one year. No other significant group differences were found over time. In all groups, however, patients showed significant improvement over time in physical activity measures. Future work is needed to explore the most effective coaching approach after an aerobic training program.

• Implications for Rehabilitation

• The active cycling program combined with education is applicable in subacute stroke patients as it required little stand-by assistance due to chip cards, the intensity was gradually built and the involvement of caregivers in the educational sessions. This training approach also revealed applicable in severely impaired stroke patients and might facilitate sport participation on the long-term.

• This randomized controlled study aims to quantify physical activity after stroke by using a combination of objective and self-report measures, which revealed detailed information about different aspects of physical activity levels.

• There is a need for coaching approaches that facilitate aerobic exercise after ending a supervised program. A coaching approach needs to guide patients in adopting aerobic exercise as a part of a lifestyle change and needs to be less time consuming.

     

Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.Influenza virus infections remain a serious seasonal health concern and the potential of severe pandemics due to the emergence of new influenza strains, such as the H5N1 “bird flu” recently identified in Hong Kong (39), provides additional impetus to develop potent and effective antiviral agents (24). At present, only amantadine and, in some countries, rimantadine are approved for the treatment and prophylaxis of influenza A infections. However, the usefulness of these two compounds is limited by their lack of activity against influenza B viruses and their rapid selection of drug-resistant mutants which remain transmissible and pathogenic (10, 25).The influenza neuraminidase, which is expressed on the virus surface, has long been considered a valid target for antiviral therapy. This enzyme, which cleaves terminal sialic acid residue from glycoconjugates, is essential for virus proliferation and infectivity (3, 17, 19, 27, 28). The observation that the structural and catalytic amino acids which line the enzyme active site are highly conserved among different influenza neuraminidase types and subtypes (reviewed in reference 6) suggests that inhibitors of this enzyme would be active against a broad range of influenza viruses.Based on information gained from crystallographic studies of influenza neuraminidases complexed with sialic acid or the transition state analog Neu5Ac2en (2, 41, 43), several potent and selective inhibitors of the influenza neuraminidases have been synthesized (15, 16, 43). Two of these, GS 4071 ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]1-cyclohexene-1-carboxylic acid), in the form of its oral prodrug GS 4104, and zanamivir (GG167, 4-guanidino-Neu5Ac2en) are currently under clinical development for the prophylaxis and treatment of influenza virus infections. Both compounds have demonstrated efficacy against influenza A and B viruses in vitro (13, 23, 40, 43, 45), in animal models of influenza virus infection (23, 31, 32, 34), and in experimental influenza virus infection in humans (11, 12, 14) when GS 4104 is taken orally and zanamivir is delivered topically to the respiratory tract as an inhalant.Although the development of resistance to zanamivir in animals or people treated with the drug has not been reported, influenza variants resistant to zanamivir due to mutations within their hemagglutinin or neuraminidase genes have been selected in vitro (1, 7, 8, 20, 38). In general, zanamivir-resistant hemagglutinin mutants have been easier to generate than neuraminidase mutations. These hemagglutinin mutants commonly contain amino acid substitutions in or near the sialic acid binding site and are believed to make the virus replication less dependent on neuraminidase activity (7, 20, 33). However, these mutations likely only affect the in vitro, not the in vivo, susceptibility to zanamivir (29).The most common neuraminidase mutation which arises in vitro under selective pressure of zanamivir has been that of the conserved Glu119 residue in the neuraminidase active site (1, 7, 38). Mutations of Glu119, which interacts with the guanidino side chain of zanamivir but not with the natural substrate (43), cause a 100-fold reduction in the sensitivity of the enzyme to zanamivir (1, 7, 38). Viruses containing mutations at this position remain infectious (8) and capable of inducing a febrile response in ferrets (5). Recently, a Lys substitution for the conserved Arg at position 292 has also been reported for a variant selected in the presence of zanamivir (8). The neuraminidase containing this mutation exhibited only a 10-fold reduction in sensitivity to zanamivir. A reassortant virus containing the mutant neuraminidase was 500-fold less infectious than wild-type virus in mice (8).In this report, we describe the first in vitro isolation and characterization of variants of a human influenza virus, A/Victoria/3/75 (H3N2), selected in the presence of GS 4071. In contrast with the experience with amantadine and rimantadine, with which drug-resistant variants can be selected after one or two passages in culture (26), variants with decreased susceptibility to GS 4071 did not readily occur. In the eighth passage, a variant containing two mutations in the stalk region of the hemagglutinin was isolated. This variant exhibited a minor decrease in susceptibility to neuraminidase inhibitors in general. A second variant, containing a conservative substitution of a Lys for an Arg at amino acid 292 of the neuraminidase enzyme active site, was isolated later in the selection process. This mutation caused a marked decrease in the susceptibility of the virus and the sensitivity of the enzyme to GS 4071. However, this mutation also adversely affected neuraminidase enzyme activity, compromised the ability of the virus to replicate in tissue culture, and reduced the infectivity of the virus 10,000-fold in mice.     

Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells

The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.     

The patients’ view: impact of the extent of resection,intraoperative imaging,and awake surgery on health-related quality of life in high-grade glioma patients—results of a multicenter cross-sectional study

Neurosurgical Review - The objective of the present study is to assess the influence of extent of resection (EoR), use of intraoperative imaging, and awake surgery on health-related quality of life...     

5-Jahres-Follow-up von 210 Columbus-Knietotalendoprothesen

Introduction

As part of 10-year documentation using the Columbus system, the interim evaluation was carried out 5 years after implantation with a collection of clinical scores, evaluation of radiological imaging and collection of statistics concerning complications.

Methods

There was a multicentre prospective recruitment of consecutive patients with the indication of implantation of a surface replacement prosthesis (Columbus CR, Deep Dish fixed inlay). Preoperatively, clinical scores were recorded (KSS and Oxford score). Five years postoperatively a new evaluation of clinical scores, the range of motion (ROM) and a radiological check-up including full leg imaging under load was performed. During this period, detailed complication documentation was made.

Results

A total of 210 patients were recruited in five centres. 187 patients were available for examination 5 years after surgery. Cumulative KSS increased from 87.5 (±26.6) preoperatively to 170 (±29.1) 5 years postoperatively. The cumulative KSS improvement was 81.5 (±35.2) points and was highly significant (p?<?0.0001; t?test). The average functional improvement in the Oxford score between the preoperative and 5?year follow-up was 21.7 (±8.8) points and was also highly significant (p?<?0.0001). The ROM improved from 106.3° (±20.2) preoperatively to 114.0° (±12.1) 5 years postoperatively (p?<?0.0001; t test). Five years after implantation, the average mechanical leg axis was 178.0° (±2.1). There was no clinically apparent or native radiologic visible aseptic loosening of the femoral or tibial joint component. There were 33 complications, but no implant-related complications such as inlay dislocation, material fracture or aseptic loosening. In total, 6 revision surgeries were performed during the follow-up period, which corresponds to a survival rate of 97.1% (CI 95%) for the implanted Columbus knee in the present patient collective for 5 years.

Conclusion

The interim analysis at 5 years of long-term observation of the Columbus system provided good clinical and radiographic results.