Pro-Inflammatory Cytokines Reduce the Proliferation of NG2 Cells and Increase Shedding of NG2 In Vivo and In Vitro

Neuron glial 2 (NG2) cells become strongly activated in injured brain areas. The activation is characterized by increased proliferation as well as increased expression and shedding of the proteoglycan NG2 expressed on their cell surface. It is currently not known how these cells respond to low-grade neuroinflammation provoked by systemic inflammation. To investigate this, we analyzed NG2 cell proliferation as well as soluble NG2 (sNG2) in cerebrospinal fluid (CSF) from rats treated with an acute intraperitoneal (i.p) injection of lipopolysaccharides (LPS) or saline and sacrificed after 2 or 24 hours. The systemically induced neuroinflammation was confirmed as elevated levels of cytokines, including interleukin (IL)-6 and IL-1β, and MHCII expressing microglia were found 24 h after LPS treatment. At this time point NG2 cell proliferation was significantly decreased in both amygdala and hippocampus and sNG2 levels in CSF were increased twofold. We also exposed human NG2 cells in culture to IL-6 and IL-1β for 24 h and found, in line with our in vivo study, a direct impact of these cytokines reducing cell proliferation and increasing shedding of NG2. We conclude that LPS induced systemic inflammation significantly affects NG2 cell proliferation and shedding and that these two events at least in in part are mediated by IL-6 and IL-1β.     

A cytochemical study of pycnotic nuclear degeneration

Pycnotic degeneration of neoplastic and normal nuclei of mice has been studied cytologically and cytochemically, in pieces of tissues removed some time after subcutaneous transplantation. The cytological changes in pycnosis were found to be about as they have often been described earlier: the nuclei become spherical, shrink progressively, the nucleoli become lost, the chromatin becomes homogeneous. The relative changes in total desoxyribose nucleic acid were followed by the Feulgen reaction; the methylgreen stain was used as an index of nuclei acid polymerization; the Millon reaction was used for the detection of the protein changes. In order to measure the amounts of the Feulgen and methylgreen dye, respectively the color developed by the Millon reaction within individual nuclei, the photometric microscopic method after Pollister and Ris was used, which allows the estimation of relative amounts of colored precipitates within individual nuclei of fixed and stained sections. Using these methods the nucleoprotein composition of resting nuclei of a viable tumortissue was compared with that of three pycnotic stages (Stage I, II, III) in nuclei of necrotic areas of the same tumor tissue (Sarcoma 180). In Sarcoma 180 the change from a fresh tumor cell to Pycnosis I involves loss of nearly half the protein, no significant decrease in desoxyribose nucleic acid, and depolymerisation of over half of the desoxyribose nucleic acid. Later these processes continue, and there is added progressive loss of DNA. It is pointed out that at any pycnotic stage there is a total protein equivalent to about 20 times the highly polymerised (methylgreen positive) DNA, which is the ratio found in non-pycnotic nuclei of the type from which these were derived. It is suggested that this part of the pycnotic chromatin represents the unaltered nucleoprotein. In transplanted liver nuclei pycnosis is much more rapid than in neoplastic tissue, but otherwise the two processes are similar both cytologically and chemically. The discussion points out that: a) there is no real increase in chromatin stainability in pycnotic nuclei; b) the change of shape in pycnosis may be due to lower nuclear viscosity accompanying the DNA depolymerisation; c) the highly polymerised state of the DNA may depend upon the presence of some particular protein, possibly histone; and d) that pyenosis can be interpreted as showing high proteolytic activity in the nucleus and a delayed nuclease activity.     

Expansion of Multipotent Stem Cells from the Adult Human Brain

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells.     

Do Cadmium, Lead, and Aluminum in Drinking Water Increase the Risk of Hip Fractures? A NOREPOS Study

The aim of this study was to investigate relations between cadmium, lead, and aluminum in municipality drinking water and the incidence of hip fractures in the Norwegian population. A trace metals survey in 566 waterworks was linked geographically to hip fractures from hospitals throughout the country (1994–2000). In all those supplied from these waterworks, 5,438 men and 13,629 women aged 50–85 years suffered a hip fracture. Poisson regression models were fitted, adjusting for age, region of residence, urbanization, and type of water source as well as other possibly bone-related water quality factors. Effect modification by background variables and interactions between water quality factors were examined (correcting for false discovery rate). Men exposed to a relatively high concentration of cadmium (IRR?=?1.10; 95 % CI 1.01, 1.20) had an increased risk of fracture. The association between relatively high lead and hip fracture risk was significant in the oldest age group (66–85 years) for both men (IRR?=?1.11; 95 % CI 1.02, 1.21) and women (IRR?=?1.10; 95 % CI 1.04, 1.16). Effect modification by degree of urbanization on hip fracture risk in men was also found for all three metals: cadmium, lead, and aluminum. In summary, a relatively high concentration of cadmium, lead, and aluminum measured in drinking water increased the risk of hip fractures, but the associations depended on gender, age, and urbanization degree. This study could help in elucidating the complex effects on bone health by risk factors found in the environment.     

Cytotoxic trans-oriented iminoether platinum complexes - Kinetics of binding to DNA oligonucleotides determined by N NMR spectroscopy

The kinetics of reactions between cytotoxic trans-oriented iminoether platinum complexes and DNA oligonucleotides have been studied by 1D and 2D [¹H, ¹⁵N] HMQC NMR spectroscopy. The results for the two isomers of the mono-iminoether compound trans-[PtCl₂(NH₃){E/Z-HNC(OMe)Me}] (trans-E and trans-Z) are compared with those of the bis-iminoether derivative trans-[PtCl₂{E-HNC(OMe)Me}₂] (trans-EE). Earlier we have shown that quite unexpectedly, trans-EE is practically inert towards a central GG residue in a 12-mer double-helical duplex. We now show that the less bulky trans-E and trans-Z compounds do bind to the interior of the duplex [5′-d(G₁G₂T₃A₄C₅C₆G₇G₈ T₉A₁₀C₁₁C₁₂)]₂ which contains terminal and central “hot” GG site. The platination by trans-E and trans-Z is as expected most pronounced for the solvent exposed, terminal GG-step but significantly, competitive binding is also observed for the central GG-step. The rate of platination of the terminal G-sites is almost an order of magnitude larger for the oligomer than for the monomer GMP which was studied for comparison. The role of trans-platinum carrier ligands in influencing the type and rate of formation of adducts with DNA and other relevant biomolecules is discussed.     

Mutational effects on transglycosylating activity of family 18 chitinases and construction of a hypertransglycosylating mutant

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.     

Ciliopathies with skeletal anomalies and renal insufficiency due to mutations in the IFT-A gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.     

Dynamic Contrast Enhanced MRI Detects Early Response to Adoptive NK Cellular Immunotherapy Targeting the NG2 Proteoglycan in a Rat Model of Glioblastoma

There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (v_e), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. v_e was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and v_e in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced v_e in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In conclusion, v_e was the most reliable radiological parameter for detecting response to intralesional NK cellular therapy.     

Consistent Condom Use Increases the Regression Rate of Cervical Intraepithelial Neoplasia 2–3

Objective

Cervical intraepithelial neoplasia grades 2-3 (CIN2-3) are usually treated by cone excision, although only 30% progress to cancer and 6–50% regress spontaneously. The aim of this study was to examine the influence of clinical factors like smoking habits, number of lifetime sexual partners, age at first sexual intercourse, sexual activity span and hormonal versus non-hormonal contraception type on the regression rate of CIN2-3.

Methods

In this prospective population-based cohort study 170 women aged 25–40 with abnormal cytology and colposcopy-directed biopsies showing first time onset CIN2-3 were consecutively included. The interval between biopsy and cone excision was standardized to minimum 12 weeks. Regression was defined as ≤CIN1 in the cone biopsy.

Results

The regression rate was 22%. Consistent condom use, defined as those women whose partners used condoms for all instances of sexual intercourse, was infrequent (n = 20, 12%). In univariate analysis consistent condom use, hormonal contraception and age at first sexual intercourse significantly predicted regression. In a multivariate analysis only consistent condom use remained as an independent predictor of regression (regression rate 55%, p = 0.001, hazard ratio = 4.4).

Conclusion

Consistent condom use between punch biopsy and cone excision in first-time onset CIN2-3 patients significantly increases the regression rate.