Serum 25-hydroxy-vitamin D3 concentrations increase during tuberculosis treatment in Tanzania.

SETTING: Vitamin D deficiency is associated with susceptibility to active tuberculosis (TB) in many settings. In vitro studies and studies on human volunteers showed that two of the first-line anti-tuberculosis drugs, isoniazid and rifampicin, reduce 25-hydroxy vitamin D (25[OH]D) concentrations. OBJECTIVE: To study changes in vitamin D status during treatment of Tanzanian hospitalised patients with pulmonary TB (PTB). DESIGN: We compared serum 25[OH]D concentrations in 81 Tanzanian PTB patients before and after 2 months of treatment. RESULTS: Median serum 25[OH]D concentrations increased from 91 nmol/l at baseline to 101 nmol/l after 2 months of TB treatment (median increase 6.0 nmol/l, IQR -0.7-25.0, P = 0.001). Median serum parathyroid hormone concentrations increased from 1.6 to 2.0 pmol/l (median increase 0.46, IQR -0.2-1.1, P < 0.001). CONCLUSION: 25[OH]D serum concentrations increased during the first 2 months of TB treatment in 81 PTB patients in northern Tanzania. Improved dietary intake and increased sunlight exposure may have contributed to the increased 25[OH]D concentrations.

[Rhinosurgery in children: developmental and surgical aspects of the growing nose]. / Rhinochirurgie bei Kindern: Entwicklungsphysiologische und chirurgische Aspekte der wachsenden Nase.

The anatomy of the nasal skeleton in newborns and adults are not alike. The complete cartilaginous framework of the neonatal nose becomes partly and gradually ossified during the years of growth and is more vulnerable to trauma in that period. Injury in the early youth may have large consequences for development of a nasal deformity which will increase during growth and reach its peak during and after the adolescent growth spurt. To understand more of the underlying problems of nasal malformations and their treatment (septoplasty) these items became the focus of multiple animal studies in the last 40 years. The effects of surgery on the nasal septum varied considerably, seemingly depending on which experimental animal was used. In review, however, it appeared that the very different techniques of surgery might be even more influential in this respect. Study of one of the larger series of experiments in young rabbits comprised skeletal measurements with statistical analysis and microscopic observations of the tissues. The behaviour of hyaline cartilage of the human nose appeared to be comparable to that of mammals. Cartilage, although resilient, can be easily fractured whereas its tendency to integrated healing is very low, even when the perichondrium has been saved. Also surgical procedures - like in septoplasty - may result in growth disturbances of the nasal skeleton like deviation or nasal spine. Loss of cartilage, as might occur after a septum abscess, is never completely restored despite some cartilage regeneration. In this article the many experimental studies are reviewed and compared. Still there remains a lack of real consensus in the literature concerning the developmental effects of rhinosurgry in children. Based on their observations in animals and a few clinical studies, mostly with small numbers of patients but with a long follow-up, the authors have compiled a list of guidelines to be considered before starting to perform surgery on the growing midface in children.

Affinity partitioning of human antibodies in aqueous two-phase systems.

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.

Low nitrification rates in acid Scots pine forest soils are due to pH-related factors.

In a previous study, ammonia-oxidizing bacteria (AOB)-like sequences were detected in the fragmentation layer of acid Scots pine (Pinus sylvestris L.) forest soils (pH 2.9-3.4) with high nitrification rates (>11.0 microg g-1 dry soil week-1), but were not detected in soils with low nitrification rates (<0.5 microg g-1 dry soil week-1). In the present study, we investigated whether this low nitrification rate has a biotic cause (complete absence of AOB) or an abiotic cause (unfavorable environmental conditions). Therefore, two soils strongly differing in net nitrification were compared: one soil with a low nitrification rate (location Schoorl) and another soil with a high nitrification rate (location Wekerom) were subjected to liming and/or ammonium amendment treatments. Nitrification was assessed by analysis of dynamics in NH4+-N and NO3- -N concentrations, whereas the presence and composition of AOB communities were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis and sequencing of the ammonia monooxygenase (amoA) gene. Liming, rather than ammonium amendment, stimulated the growth of AOB and their nitrifying activity in Schoorl soil. The retrieved amoA sequences from limed (without and with N amendment) Schoorl and Wekerom soils exclusively belong to Nitrosospira cluster 2. Our study suggests that low nitrification rates in acidic Scots pine forest soils are due to pH-related factors. Nitrosospira cluster 2 detected in these soils is presumably a urease-positive cluster type of AOB.

Presence of Nitrosospira cluster 2 bacteria corresponds to N transformation rates in nine acid Scots pine forest soils.

The relation between environmental factors and the presence of ammonia-oxidising bacteria (AOB), and its consequences for the N transformation rates were investigated in nine Scots pine (Pinus sylvestris L.) forest soils. In general, the diversity in AOB appears to be strikingly low compared to other ecosystems. Nitrosospira cluster 2, as determined by temporal temperature gradient electrophoresis and sequencing, was the only sequence cluster detected in the five soils with high nitrification rates. In the four soils with low nitrification rates, AOB-like sequences could not be detected. Differences in nitrification rates between the forest soils correlated to soil C/N ratio (or total N) and atmospheric N deposition.

Biodiversity effects on soil processes explained by interspecific functional dissimilarity.

The loss of biodiversity can have significant impacts on ecosystem functioning, but the mechanisms involved lack empirical confirmation. Using soil microcosms, we show experimentally that functional dissimilarity among detritivorous species, not species number, drives community compositional effects on leaf litter mass loss and soil respiration, two key soil ecosystem processes. These experiments confirm theoretical predictions that biodiversity effects on ecosystem functioning can be predicted by the degree of functional differences among species.

Indications for the tracking of elevated nitrogen levels through the fungal route in a soil food web.

The objective of the present study was to determine the effects of elevated N in dead organic matter on the growth of fungi and to establish the consequences for the development of microbivores. Therefore, three fungal species were cultured on Scots pine litter differing in N content. The growth of the soil fungal species Trichoderma koningii, Penicillium glabrum and Cladosporium cladosporioides was directly influenced by the N content (ranging from 1.25 to 2.19% N) of the substrate. For all three fungal species maximum growth was highest at intermediate N content (1.55%) of the substrate. The fungivorous collembolan Orchesella cincta reached highest asymptotic body mass when fed with C. cladosporioides, grown on litter medium with intermediate N content (1.55%). The growth of O. cincta was lower when fed with C. cladosporioides from litter medium with the highest N content (2.19%). Similar results were obtained in mesocosm experiments in which pine litter with three levels of N (1.11, 1.78, 2.03% N) was used as substrate for the fungi. On litter with the highest N content (2.03%) hyphal length and asymptotic body mass of O. cincta were reduced. The results show that the N content of the substrate determines the growth of both fungi and fungivores, and suggest that elevated levels of N in soil track through the fungal part of the soil food web.

Subglottic stenosis after endolaryngeal intubation in infants and children: result of wound healing processes.

OBJECTIVE: To study the histopathology of subglottic stenosis in children of different ages after treatment during different periods of time, with or without laser application. Partial resection of the anterior cricoid with adhering stenotic subglottic area in the live young patient provides unique material for studying wound healing and scarring processes. METHODS: 25 specimens obtained from partial cricotracheal resection (PCTR) in children, were histologically processed and stained with Haematoxylin and Eosin, Resorcin and Fuchsin (for elastic fibers), and immunohistochemical staining (for the presence of macrophages). RESULTS: All specimens were found to have severe and sclerotic scarring with squamous metaplasia of the epithelium, loss of glands and elastic mantle fibers (tunica elastica), and dilation of the remaining glands with formation of cysts. Also, the cricoid cartilage was affected on the internal and external side, with irreversible loss of perichondrium on the inside and resorption by macrophages of cartilage on both sides. Detrimental effects of laser therapy were demonstrated in four cases. The normal intercellular matrix was completely destroyed and the number of chondrocytes in the cartilage structure diminished. CONCLUSION: Wound healing after laryngeal injury is a process of intense restoration and reorganization of the various tissues involved. This process, however, does not guarantee complete repair. In the severe cases irreversible scarring has replaced normal tissues. There seems to be no direct relationship between the length of the post-lesional period, the age of the patient and the severity of the stenosis. When subglottic stenosis has developed and the majority of the tissues is replaced by dense fibrous tissue, PCTR is strongly indicated to achieve renewed patency of the airway.

Serum transferrin receptor concentration indicates increased erythropoiesis in Kenyan children with asymptomatic malaria.

BACKGROUND: Serum transferrin receptor concentrations indicate both erythropoietic activity and the deficit of functional iron in the erythron. In contrast with serum ferritin concentrations, serum transferrin receptor concentrations are not or are only marginally influenced by the inflammatory response to infection. OBJECTIVE: We assessed iron status and examined the relation between serum transferrin receptor concentrations and malaria in children aged 2-36 mo who were asymptomatic for malaria. DESIGN: This was a community-based cluster survey (n = 318). RESULTS: Prevalences of malaria, anemia (hemoglobin concentration <110 g/L), iron deficiency (serum ferritin concentration <12 microg/L), and iron deficiency anemia were 18%, 69%, 53%, and 46%, respectively. Malaria was associated with lower mean hemoglobin concentrations (92.7 compared with 104.1 g/L; P = 0.0001) and higher geometric mean serum concentrations of transferrin receptor (11.4 compared with 7.8 mg/L; P = 0.005), ferritin (21.6 compared with 11.9 microg/L; P = 0.05), and C-reactive protein (12.5 compared with 6.8 mg/L; P = 0.004). There was no evidence for an association between serum concentrations of C-reactive protein and transferrin receptor. Children with malaria had higher serum transferrin receptor concentrations than expected for the degree of anemia, even after adjustment for inflammation indicated by serum C-reactive protein concentration quartiles (P = 0.02). CONCLUSIONS: Our findings are consistent with the notion that malaria-induced hemolysis is accompanied by increased erythropoiesis. Serum transferrin receptor concentration is not useful for detecting iron deficiency in individuals with malaria. Individuals with high concentrations of serum C-reactive protein or similar acute phase reactants should be excluded from analysis if serum ferritin concentrations <12 microg/L are to be used to measure iron deficiency in malaria-endemic areas.

The potency of culture-expanded nasal septum chondrocytes for tissue engineering of cartilage.

Tissue engineering techniques to create extra autologous cartilage for reconstructive surgery receive more and more scientific and industrial attention. The objective of this experimental study was to assess the use of in vitro multiplied chondrocytes of the nasal septum for generation of cartilage grafts using tissue engineering techniques. Cells isolated from a biopsy of septal cartilage of rabbits and humans were expanded in culture to get a sufficient number of cells to engineer a cartilage graft. The drawback of the expansion procedure is that the cells lose their cartilaginous phenotype (dedifferentiation). We studied a method to reverse the dedifferentiation of expanded cells to stimulate them to produce cartilage matrix of good quality. Rabbit chondrocytes showed reversion of dedifferentiation (redifferentiation) when fetal calf serum was replaced by the growth factors IGF1 and TGFbeta2. This was expressed by increased glycosaminoglycan synthesis and increased numbers of collagen type II-producing cells. The redifferentiation capacity of septal cartilage cells of young rabbits was higher than that of adult rabbits. In human chondrocytes from the nasal septum redifferentiation could also be induced by replacement of serum with IGF1 and TGFbeta2. This method, however, was less efficient than in rabbits. Chondrocytes of older patients (>40 years old) were no longer sensitive to the growth factor treatment. In conclusion, our study demonstrates a method to regain cartilage phenotype in multiplied cells of nasal septum cartilage needed for tissue engineering of new cartilage. These results are promising for this technique to generate cartilage grafts for facial plastic surgery of the nasal septum.

Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model.

OBJECTIVE: The ability of cartilage to regenerate following injury is limited, potentially leading to osteoarthritis. Integrative cartilage repair, necessary for durable restoration of cartilage lesions, can be regarded as a wound healing process. Little is known about the effects of growth factors regulating acute cartilage wound healing in vivo. In this study the temporal expression patterns of growth factors and proteoglycan content in cartilage wound edges in vivo were studied. DESIGN: Cartilage wounds were created in rabbit ear cartilage using a 6 mm biopsy punch. Specimens were subsequently harvested 1, 3, 7, 14 and 28 days after surgery. Paraffin sections were thionin stained to visualize proteoglycan loss and replacement. Immunohistochemical staining of TGFbeta1, TGFbeta3, IGF-1, IGF-II and FGF-2 was used to define growth factor expression at the cartilage wound sites. RESULTS: Almost no effect of cartilage wounding was observed one day after surgery. A decrease of proteoglycan content, with a maximal loss at day 7, and a subsequent restoration was observed at the wound edges. Growth factor expression increased simultaneously. Maximal immunostaining for IGF1, IGFII, FGF2 and TGF-beta3 was observed at day 7, followed by a gradual decrease. Increased expression of TGFbeta1 lasted from day 3 until day 14. CONCLUSION: We have demonstrated the ability of chondrocytes to increase growth factor expression and to restore the rapid decrease in proteoglycan content in the initial phase following acute wounding. A temporal increase in intracellular growth factor expression suggests an autocrine and/or paracrine metabolic stimulation, which can be regarded a sign of chondrocytes repair capacity.

A new in vivo model for testing cartilage grafts and biomaterials: the 'rabbit pinna punch-hole' model.

In this study an animal model was developed for evaluation of the feasibility of cartilage grafts. In the cartilage of the external ear of the rabbit multiple holes, 6 mm in diameter, were punched, leaving the adherent skin intact. Different experimental groups were evaluated. First, the punch-hole model was validated under various conditions to study spontaneous or perichondrial initiated regeneration of the cartilage defect. When both cartilage and perichondrium was excised no spontaneous repair of the cartilage defect was observed. When perichondrium is present, variable patch-like closure of the punch hole was found. As 'golden standard' a punched out piece of cartilage was reimplanted directly. This condition showed adequate closure of the punch hole, however, no perfect integration of graft and surrounding cartilage was observed. Secondly, to evaluate the 'punch-hole model' a biomaterial, trabecular demineralized bovine bone matrix (DBM), was implanted and tested as a scaffold for tissue engineering techniques in vivo and in vitro. Direct implantation of DBM did not lead to any cartilage formation to close the defect. In vivo engineered cartilage, generated by enveloping DBM in perichondrium for 3 weeks, could adequately close the punch hole. When DBM was seeded with isolated chondrocytes in vitro before implantation in the defect, a highly fragmented graft, with some islets of viable cells was seen. To promote an efficient and reliable evaluation of cartilage grafts a semi-quantitative grading system was developed. Items such as quality, quantity and integrity of the cartilage graft were included in a histomorphological grading system to provide information about the properties of a specific cartilage graft. To validate the grading system, all conditions were scored by two independent observers. An excellent reliability (R = 0.96) was seen between the observers. In summary, the rabbit pinna punch-hole model is a reliable and efficient method for first evaluation of cartilage grafts. The results can be easily analyzed using a semi-quantitative grading system.

In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction.

To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.

Spatiotemporal Stability of an Ammonia-Oxidizing Community in a Nitrogen-Saturated Forest Soil.

Elevated levels of nitrogen input into various terrestrial environments in recent decades have led to increases in soil nitrate production and leaching. However, nitrifying potential and nitrifying activity tend to be highly variable over space and time, making broad-scale estimates of nitrate production difficult. This study investigates whether the high spatiotemporal variation in nitrate production might be explained by differences in the structure of ammonia-oxidizing bacterial communities in nitrogen-saturated coniferous forest soils. The diversity of ammonia-oxidizing bacteria of the b-subgroup Proteobacteria was therefore investigated using two different PCR-based approaches. The first targeted the 16S rRNA gene and involved temporal temperature gradient electrophoresis (TTGE) of specifically amplified PCR products, with subsequent band excision and nucleotide sequence determination. The second approach involved the cloning and sequencing of PCR-amplified amoA gene fragments. All recovered 16S rDNA sequences were closely related to the culture strain Nitrosospira sp. AHB1, which was isolated from an acid soil and is affiliated with Nitrosospira cluster 2, a sequence group previously shown to be associated with acid environments. All amoA-like sequences also showed a close affinity with this acid-tolerant Nitrosospira strain, although greater sequence variation could be detected in the amoA analysis. The ammonia-oxidizing bacterial community in the nitrogen-saturated coniferous forest soil was determined to be very stable, showing little variation between different organic layers and throughout the year, despite large differences in the total Bacterial community structure as determined by 16S rDNA DGGE community fingerprinting. These results suggest that environmental heterogeneity affecting ammonia oxidizer numbers and activity, and not ammonia oxidizer community structure, is chiefly responsible for spatial and temporal variation in nitrate production in these acid forest soils.

Langerhans cell histiocytosis of the larynx.

A pediatric case of Langerhans cell histiocytosis leading to severe and recurrent subglottic stenosis, ultimately necessitating partial cricotracheal resection, is presented and the literature on this very rare disorder is briefly reviewed.

Fixation-dependent immunolocalization shift and immunoreactivity of intracellular growth factors in cartilage.

The effects of fixation on immunolocalization and immunoreactivity in cartilage tissues were studied using monoclonal antibodies against peptides that can effectively stimulate chondrocytes in vitro and have been shown to play a role in musculoskeletal tissue regeneration: transforming growth factor beta1, transforming growth factor beta3, insulin-like growth factor I, insulin-like growth factor II and fibroblast growth factor 2. Paraffin sections fixed in buffered formalin, buffered paraformaldehyde, Carnoy and methacarn, as well as cryosections, were tested. A strong immunoreaction was observed in tissue fixed in formaldehyde-based fixatives, with a resemblance to that in cryopreserved tissues. Immunoreactivity was reduced in alcohol-fixed tissues. Furthermore, a striking intracellular immunolocalization shift from cytoplasm to nucleus was observed using alcohol-based fixatives as compared to cryopreserved or formaldehyde-based fixatives. We concluded that, for the detection and localization of growth factors in cartilage tissues, fixation in buffered formalin or paraformaldehyde is optimal.

Chondrogenic potential of in vitro multiplied rabbit perichondrium cells cultured in alginate beads in defined medium.

Perichondrium has a chondrogenic capacity and is therefore a candidate tissue for engineering of cartilage in vitro. Donor age and culture conditions probably influence chondrogenesis. The aim of this study was to compare the chondrogenic capacity of ear and nasal perichondrium from young and adult rabbits, using serum containing and serum-free culture conditions. This study demonstrates that more than 1 million cells can be generated out of 1 cm(2) of perichondrium tissue in 3-5 weeks of culture, irrespective of age. Culturing of these cells in alginate in medium with 2, 10, or 20% fetal calf serum did result in the production of small amounts of glycosaminoglycan, but no collagen type II was demonstrated. When serum was replaced however by insulin-like growth factor-1 (IGF-1) (10 ng/mL) plus transforming growth factor-beta2 (TGF-beta2) (10 ng/mL) an increased glycosaminoglycan production and induction of collagen type II was found, especially in cells isolated from perichondrium of the ear. Cells derived from perichondrium of young rabbits showed larger chondrogenic potential than cells from perichondrium of adult rabbits. Moreover, stimulation of both glycosaminoglycan synthesis and collagen type II production was about five times higher in cells isolated from the ear perichondrium of young rabbits than of adult rabbits. We conclude that young auricular perichondrium seems a useful source of cells for tissue engineering of cartilage when cultured in serum-free medium in combination with IG-F1 and TGF-beta2.

Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM).

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.