Complete regeneration of large bone defects in rats with commercially available fibrin loaded with BMP-2.

This study aimed at investigating in vitro and in vivo the efficiency of commercially available fibrin as a carrier for controlled and sustained bone morphogenetic protein-2 (BMP-2) release to induce bone formation and reduce the side effects of its use. In vitro release and activity of low-dose recombinant human BMP-2 (rhBMP-2) (37.5 µg/mL) embedded in commercially available fibrin were evaluated and, subsequently, critical-size femur defects in rats were grafted to study bone regeneration and vascularisation by micro-computed tomography (µCT) and histology. In vitro experiments showed a sustained BMP-2 release with a high BMP activity remaining after 28 d. In vivo, fibrin loaded with BMP-2 showed an extremely fast bone healing, with a large amount of new bone formation throughout the entire defect in the first 4 weeks and complete cortical repair and fusion after 8 weeks, with no ectopic bone formation. In contrast, the control fibrin group did not fuse after 12 weeks. Vascularisation was similar in both groups at 4 and 12 weeks after implantation. In conclusion, commercially available fibrin is a very efficient carrier for rhBMP-2 to graft critical-size cortical bone defects and might be a more optimal delivery vehicle for BMP-2-induced bone regeneration than currently available collagen sponges.

Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels.

Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2). This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting load-bearing defects in trauma and orthopaedic surgery.

Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defects.

Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.

Patients' representations of their end-stage renal disease: relation with mortality.

BACKGROUND: Self-regulation theory explains how patients' illness perceptions influence self-management behaviour (e.g. via adherence to treatment). Following these assumptions, we explored whether illness perceptions of ESRD-patients are related to mortality rates. METHODS: Illness perceptions of 182 patients participating in the NECOSAD-2 study in the period between December 2004 and June 2005 were assessed. Cox proportional hazard models were used to estimate whether subsequent all-cause mortality could be attributed to illness perception dimensions. RESULTS: One-third of the participants had died at the end of the follow-up. Mortality rates were higher among patients who believed that their treatment was less effective in controlling their disease (perceived treatment control; RR = 0.71, P = 0.028). This effect remained stable after adjusting for sociodemographic and clinical variables (RR = 0.65, P = 0.015). CONCLUSIONS: If we consider risk factors for mortality, we tend to rely on clinical parameters rather than on patients' representations of their illness. Nevertheless, results from the current exploration may suggest that addressing patients' personal beliefs regarding the effectiveness of treatment can provide a powerful tool for predicting and perhaps even enhancing survival.

Pleural drainage and pleurodesis: implementation of guidelines in four hospitals.

The aim of the present study was to evaluate the implementation of the 2003 Dutch guideline on the diagnosis and treatment of malignant pleural effusions, and the potential effect of the implementation on the clinical outcome of pleurodesis. All patients with malignant pleural effusion who had a pleural drain placed with the intention of performing pleurodesis were registered prospectively in four centres. Details of the procedure and fluid recurrence and survival data were noted. Patients with a proven malignancy (n = 100) were entered into the registration database. Diagnostic guideline recommendations were followed in 60-70% of the patients. Surprisingly, pleurodesis was performed in only 75% of the patients, mainly due to the presence of a trapped lung. All pleurodeses were performed using talc, according to the guideline. Follow-up revealed fluid recurrence in 27 (36%) patients after a mean follow-up of 17 days (range 2-285 days); 14 patients with successful pleurodesis died with a median survival of 61 days (range 13-174 days). Systemic treatment following pleurodesis and good apposition of the pleural surfaces during drainage were good prognostic factors. Despite reasonable-to-good adherence to the guideline, the number of successful pleurodeses was low. Better predictors of a good pleurodesis outcome are needed.

Basic helix-loop-helix transcription factor profiling of lung tumors shows aberrant expression of the proneural gene atonal homolog 1 (ATOH1, HATH1, MATH1) in neuroendocrine tumors.

Microarray-based expression profiling studies of lung adenocarcinomas have defined neuroendocrine subclasses with poor prognosis. As neuroendocrine development is regulated by members of the achaete-scute and atonal classes of basic helix-loop-helix (bHLH) transcription factors, we analyzed lung tumors for expression of these factors. Out of 13 bHLH genes tested, 4 genes, i.e., achaete-scute complex-like 1 (ASCL1, HASH1, Mash1), atonal homolog 1 (ATOH1, HATH1, MATH1), NEUROD4 (ATH-3, Atoh3, MATH-3) and neurogenic differentiation factor 1 (NEUROD1, NEUROD, BETA2), showed differential expression among lung tumors and absent or low expression in normal lung. As expected, tumors that have high levels of ASCL1 also express neuroendocrine markers, and we found that this is accompanied by increased levels of NEUROD1. In addition, we found ATOH1 expression in 9 (16%) out of 56 analyzed adenocarcinomas and these tumors showed neuroendocrine features as shown by dopa decarboxylase mRNA expression and immunostaining for neuroendocrine markers. ATOH1 expression as well as NEUROD4 was observed in small cell lung carcinoma (SCLC), a known neuroendocrine tumor. Since ATOH1 is not known to be involved in normal lung development, our results suggest that aberrant activation of ATOH1 leads to a neuroendocrine phenotype similar to what is observed for ASCL1 activation during normal neuroendocrine development and in lung malignancies. Our preliminary data indicate that patients with ATOH1-expressing adenocarcinomas might have a worse prognosis.

Acute pulmonary histoplasmosis from Ghana.

A male patient developed acute pulmonary histoplasmosis 2 weeks after bathing in the water falls of Wli, Ghana. Exposure to Histoplasma capsulatum was probably mediated through inhalation of an aerosol of water and guano from the large colony of fruit bats of the falls. More cases of acute pulmonary histoplasmosis can be expected.

Pleural thickening in a construction worker: it is not always mesothelioma.

We describe the case of a 45-year-old man presenting with chest pain and pleural effusions. These symptoms were progressive over a period of three years, with pericardial involvement and respiratory insufficiency finally resulting in death. Despite repeated diagnostic procedures, a final diagnosis could only be made at autopsy. Multisystem foamy histiocyte infiltration suggested the diagnosis of Erdheim-Chester disease.

Occupational asbestos exposure: how to deal with suspected mesothelioma cases--the Dutch approach.

INTRODUCTION: Patients with asbestos-related diseases, such as malignant mesothelioma (MM), are not uniformly treated in Europe when they apply for compensation. In The Netherlands, the Institute of Asbestos Victims (IAV) acts on behalf of patients with a malignant mesothelioma. In the majority of cases, the diagnosis is clear but in some, uncertainty remains. In these cases a specialist opinion of the Mesothelioma Group of the Dutch Thoracic Society (DTS) is required. The process of data handling and final outcome for these patients is discussed and compared with the situation in other European countries. MATERIALS AND METHODS: Dutch patients with a possible malignant mesothelioma and occupational exposure to asbestos presented their cases to the IAV. In 10% of the cases, pathological confirmation of a malignant mesothelioma could not be obtained. These cases were presented to the Mesothelioma Group to obtain a clinical diagnosis based on clinical reports, occupational history, X-ray examination and other factors. Each case was reviewed by three independent pulmonologists experienced in MM. The majority view was binding for acceptance or rejection of the diagnosis. RESULTS: In the period January 2000 until May 2005, the IAV received 1747 cases for compensation. In 161 cases no definitive diagnosis could be made on pathology and were presented to the Mesothelioma Group. Of these cases, 117 (73%) were considered to be compatible with the clinical diagnosis malignant pleural mesothelioma. Forty-four cases (27%) were rejected. In 75% of the cases (112 of 150), the conclusion of the three independent specialists was unanimous; in 11 cases one specialist refrained from a diagnosis. The median time from request to submission of the report was 34 days (range 1-185 days). CONCLUSIONS: Compared with other European countries, this approach, as determined by the IAV and Mesothelioma Group of the DTS, is an effective and rapid way to investigate claims of patients with a possible occupationally related malignant mesothelioma.

Th1/Th2 cytokine imbalance in a family with hyper-IgE syndrome.

BACKGROUND: Hyperimmunoglobulin E (hyper-IgE) syndrome is a rare immunodeficiency characterised by recurrent skin and respiratory tract infections, skeletal and dental abnormalities, chronic eczema, and elevated serum IgE. We describe a family with four hyper-IgE syndrome patients (38, 37, 30 and 7 years old), in which we investigated the cytokine response to both specific and non-specific stimulation. METHODS: Whole blood from patients and volunteers was stimulated for either 24 or 48h at 37 degrees C with heat-killed Staphylococcus, C. albicans or a combination of IL-12 and IL-18. Cytokine concentrations in the plasma were measured by specific radioimmuno-assays or ELISA. RESULTS: Serum IgE ranged from 5,000 to 16,670 IU/ml, and neutrophil chemotaxis was normal in all four patients. Tumour necrosis factor, interleukin (IL)-1beta, IL-6 and IL-8 production after stimulation of whole-blood cultures with lipopolysaccharide or heat-killed S. aureus did not differ between the adult patients and four healthy controls. In contrast, when blood from patients and controls was stimulated with heat-killed S. aureus or C. albicans, a severe imbalance towards a Th2 phenotype was found, with 10- to 30-fold reduction in the IFNgamma/IL-10 ratios in the hyper-IgE syndrome patients. The IFNgamma production in the patients was less severely impaired when blood was non-specifically stimulated with a combination of IL-18 and IL-12. CONCLUSION: In this family with hyper-IgE syndrome, the imbalance in the Th1/Th2 cytokine production may have been involved in the pathogenesis of the recurrent infections and/or chronic eczema characteristic of this disease.

Toxicity of high-dose radiotherapy combined with daily cisplatin in non-small cell lung cancer: results of the EORTC 08912 phase I/II study. European Organization for Research and Treatment of Cancer.

The purpose of this work was to study the feasibility of concurrent chemoradiation in patients with inoperable non-small cell lung cancer (NSCLC). 40 patients with inoperable NSCLC were treated with escalating doses of radiotherapy and cisplatin (cDDP). The radiation dose was increased step by step from 60.5 to 66 Gy in daily fractions of 2.75 Gy. Chemotherapy was also increased step by step from 20 to 24 daily doses of cDDP 6 mg/m(2) and given concurrently with radiotherapy. A dose of 40 Gy/2 Gy/20 fractions (fx) was given to the EPTV (elective planning target volume) which included the gross tumour volume with a margin of 2 cm and part of or the entire mediastinum. During each session a boost dose of 0.75 Gy was given simultaneously to the BPTV (boost planning target volume), which encompassed the GTV (gross tumour volume) with a margin of 1 cm, for the first 20 fx, so the total dose to the tumour was 55 Gy. Cisplatin 6 mg/m(2) was given 1 h prior to radiotherapy at each fraction. From then on the dose of radiation to the BPTV and the dose of cDDP were increased step by step. In group I the BPTV was irradiated with two extra fractions of 2.75 Gy to a total dose of 60. 5 Gy without cDDP. In group II the same total dose of 60.5 Gy was given but the last two fractions were combined with cDDP. In group III four extra fractions of 2.75 Gy were given to the BPTV to a total dose of 66 Gy, only two of these fractions combined with cDDP. Finally, in group IV a total dose of 66 Gy was given in 24 fractions, all fractions combined with cDDP. All patients were planned by means of a CT-based conformal treatment planning. The maximal length of the oesophagus receiving >/=60.5 Gy was 11 cm. 40 patients were evaluable for acute and late toxicity and for survival. Acute toxicity grade >/=3 (common toxicity criteria, CTC) was rarely observed; nausea/vomiting in 3 patients (8%), leucopenia in 2 patients (5%), thrombocytopenia in 2 patients (5%), whilst 2 patients (5%) suffered from severe weight loss. Late side-effects (European Organization for Research and Treatment of Cancer/Radiation Therapy Oncology Group, EORTC/RTOG) were: oesophageal toxicity >/=grade 3 in 2 patients (5%) and radiation pneumonitis grades 1 (3%) and 2 (3%) in 1 patient each. Overall actuarial 1- and 2-year survival was 53% and 40%, respectively. The 1- and 2-year local disease-free interval was 65% and 58% respectively. Radiotherapy at a dose of 66 Gy/2.75 Gy/24 fx combined with daily cDDP 6 mg/m(2) given over 5 weeks is feasible and results in a good local disease-free interval and a good survival rate. This treatment schedule is at present being tested as one of the two treatment arms of EORTC phase III study protocol 08972/22973.

Fibrillary-immunotactoid glomerulopathy with renal deposits of IgAlambda: a rare cause of glomerulonephritis.

We describe a 24-year-old patient who presented with a nephrotic syndrome. His renal biopsy revealed a diffuse mesangioproliferative glomerulonephritis with eosinophilic deposits. Electron microscopy showed organized, Congo-red negative deposits, forming microtubules of about 20 nm width in the capillary walls and in the mesangium, establishing a diagnosis of fibrillary-immunotactoid glomerulopathy. Fibrillary-immunotactoid glomerulopathy is a rare cause of glomerulonephritis, characterized by Congo-red-negative glomerular deposits of fibrils, sometimes organized in microtubules, predominantly containing IgG and C3. Patients clinically present with the nephrotic syndrome, hematuria and hypertension. The pathogenesis of this glomerulopathy has not been elucidated yet. In our patient, the renal deposits contained IgAlambda. This peculiar feature is suggestive of an underlying paraproteinemia. However, in the serum no paraproteins or cryoglobulins were found, and also microscopical examination and immunophenotyping of the bone marrow did not point to the presence of a monoclonal plasma cell dyscrasia. Our patient was not treated with immunosuppressive drugs and he is currently progressing to end-stage renal disease.

Pharmacokinetic/pharmacodynamic modelling of the eosinopenic and hypokalemic effects of formoterol and theophylline combination in healthy men.

Interactions of formoterol and theophylline were evaluated with the use of pharmacokinetic-pharmacodynamic (PK/PD) modelling. Oral doses of 144 microg of formoterol and 375 mg of theophylline were given separately or combined to healthy subjects. As effect parameters, plasma eosinophil and potassium concentrations were used. Kinetic interactions between formoterol and theophylline were not found. Plasma drug concentrations were linked to the observed effects via an effect compartment model with a sigmoid E max model. The E max values+/-SD for the hypokalemic effects were 2.29+/-0.78 mmol/l for formoterol and 1.64+/-1.16 mmol/l for theophylline (P>0.05). The E max values for the eosinopenic effects were fixed at zero. The EC 50 values of the eosinopenic and hypokalemic effects were respectively 91.4+/-38.2 pg/ml and 128.4+/-52.9 pg/ml for formoterol, and 11. 9+/-4.6 microg/ml and 15.5+/-4.8 microg/ml for theophylline. Effects of both drugs combined were described with a non-competitive interaction model. The correlation coefficients of the fits of the eosinopenic and hypokalemic effects were respectively 0.9520+/-0. 0311 and 0.9371+/-0.0227, supporting our hypothesis of non-competitive interaction.

[Ocular infection by Pseudomonas aeruginosa in a mechanically ventilated patient]. / Ooginfectie door Pseudomonas aeruginosa bij een mechanisch beademde patiënt.

A 59-year-old man developed bilateral keratitis several weeks after the initiation of mechanical ventilation because of respiratory failure and sepsis following abdominal surgery. Colonisation of the upper airways by P. aeruginosa had been established before. Invasion through corneal epithelial defects based on dehydration keratitis was the presumed route of infection. Despite aggressive treatment, including antibiotics, the infection was rapidly progressive in both eyes. The patient died of deterioration of his general condition. In order to prevent such eye infections in a patient on mechanical ventilation, there is a need of good eye care, prevention of corneal lesions and alertness, especially when the patient is colonised by virulent micro-organisms like P. aeruginosa.

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.

Optimizing screening procedures for Staphylococcus aureus nasal carriage in patients on haemodialysis.

BACKGROUND: So far it remains unclear what the optimal screening method for detection of S. aureus nasal carriage in patients on haemodialysis is with regard to number of cultures performed, culture interval, and necessity of a broth-enrichment procedure. METHODS: A prospective, uncontrolled study was performed at the renal unit of a tertiary care centre, including all haemodialysis patients (n=91) attending the unit during the study period. The purpose was to determine the optimal screening method for S. aureus nasal carriage in patients on haemodialysis. RESULTS: When compared to the conventional culture method, inclusion of a broth-enrichment procedure increased the number of cultures positive for S. aureus significantly (31 vs 24%, P<0.0001). Of 91 patients 37% were S. aureus carriers (defined as at least 1 of 5 cultures positive), 33% were stable carriers (defined as at least 2 of 5 cultures positive). Fourth and 5th cultures, taken at subsequent dialysis sessions, captured only two additional carriers (6% of all carriers). With respect to culture results and identification of carrier status a short (1-h) and a long (>24-h) sampling procedure showed no significantly different results. CONCLUSIONS: S. aureus nasal carriage in haemodialysis patients can be conveniently established with three nasal cultures taken with 1-h intervals, and the inclusion of a broth-enrichment procedure.