Biomechanical evaluation of graded ventralfacetectomy simulating foraminoplasty of percutaneous endoscopic lumbar discectomy.

OBJECTIVE: To explore the lumbar spine biomechanics of graded ventral facetectomy and determine the appropriate extent of resection for foraminoplasty. PATIENTS AND METHODS: We retrospectively measured several radiological parameters of superior articular process (SAP) and bony intervertebral foramen in computed tomography scans of 170 lumbar vertebral discs. The intact finite element (FE) spine of L2-sacrum was modified to simulate foraminoplasty with two typical graded ventral facetectomy methods (Method Ⅰ: basal part resection of SAP; Method Ⅱ: apical part resection of SAP) to explore the biomechanical effects under different physiological motions. RESULTS: Examination of the radiological parameters of the bony intervertebral foramen indicated that they were generally narrower than the diameters of commercially available working cannulas. Some of these parameters showed gender differences. The biomechanical evaluation indicated that the range of motion increased gradually with the expansion of the resection extent, and the differences compared to the intact spine at the same level were greater in Method I than in Method Ⅱ. CONCLUSIONS: The appropriate ventral resection extent of the basal part of the SAP (Method I) was 4 mm, 3 mm, and 3 mm on the lateral view at L3-L4, L4-L5, and L5-S1, respectively. The appropriate ventral resection extent of the apical part of the SAP (Method II) were 10 mm, 6 mm and 6 mm on the lateral view at L3-L4, L4-L5, and L5-S1, respectively. Extensive resection of foraminoplasty may destabilize lumbar motion segments.

[Efficacy analysis of anti-thymocyte globulin regimens with different timing strategies for matched sibling donor hematopoietic stem cell transplantation].

Objective: To compare the effects of two administration time strategies for rabbit antihuman thymocyte immunoglobulin (rATG) of 5mg/kg total dose in matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) . Methods: This study retrospectively analyzed the clinical data of 32 patients who received MSD-HSCT with 5 mg/kg rATG conditioning regimen at the Department of Hematology of the First Medical Center of the People's Liberation Army General Hospital from October 2020 to April 2022. The patients were classified into two groups: the 4d-rATG group (16 cases), who received antithymocyte globulin (ATG) from day -5 to day -2, and the 2d-rATG group (16 cases), who received ATG from day -5 to day -4. Between the two groups, the transplantation outcomes, serum concentrations of active antithymocyte globulin (ATG) in patients from -4 days to 28 days after graft infusion (+28 days), and the reconstitution of lymphocyte subsets on days +30, +60, and +90 were compared. Results: The cumulative incidences of acute graft-versus-host disease at 100 days after graft infusion were 25.0% (95% CI 7.8% -47.2% ) and 18.8% (95% CI 4.6% -40.2% ) (P=0.605) in the 4d-rATG group and 2d-rATG group, respectively. The 1-year cumulative incidences of chronic graft-versus-host disease were 25.9% (95% CI 8.0% -48.6% ) and 21.8% (95% CI 5.2% -45.7% ) (P=0.896). The 1-year cumulative incidence of relapse was 37.5% (95% CI 18.9% -65.1% ) and 14.6% (95% CI 3.6% -46.0% ) (P=0.135), and the 1-year probabilities of overall survival were 75.0% (95% CI 46.3% -89.8% ) and 100% (P=0.062). The total area under the curve (AUC) of serum active ATG was 36.11 UE/ml·d and 35.89 UE/ml·d in the 4d-rATG and 2d-rATG groups, respectively (P=0.984). The AUC was higher in the 4d-rATG group than that in the 2d-rATG group (20.76 UE/ml·d vs 15.95 UE/ml·d, P=0.047). Three months after graft infusion, the average absolute count of CD8(+) T lymphocytes in the 4d-rATG group was lower than that in the 2d-rATG group (623 cells/µl vs 852 cells/µl, P=0.037) . Conclusion: The efficiencies of GVHD prophylaxis in MSD-PBSCT receiving 4d-ATG regimen and the 2d-rATG regimen were found to be similar. The reconstruction of CD8(+)T lymphocytes in the 2d-rATG group was better than that in the 4d-rATG group, which is related to the lower AUC of active ATG after transplantation.

Pathological missense mutations of neural cell adhesion molecule L1 affect homophilic and heterophilic binding activities.

Mutations in the gene for neural cell adhesion molecule L1 (L1CAM) result in a debilitating X-linked congenital disorder of brain development. At the neuronal cell surface L1 may interact with a variety of different molecules including itself and two other CAMs of the immunoglobulin superfamily, axonin-1 and F11. However, whether all of these interactions are relevant to normal or abnormal development has not been determined. Over one-third of patient mutations are single amino acid changes distributed across 10 extracellular L1 domains. We have studied the effects of 12 missense mutations on binding to L1, axonin-1 and F11 and shown for the first time that whereas many mutations affect all three interactions, others affect homophilic or heterophilic binding alone. Patient pathology is therefore due to different types of L1 malfunction. The nature and functional consequence of mutation is also reflected in the severity of the resultant phenotype with structural mutations likely to affect more than one binding activity and result in early mortality. Moreover, the data indicate that several extracellular domains of L1 are required for homophilic and heterophilic interactions.

Alternative splicing of human NrCAM in neural and nonneural tissues.

Neural cell adhesion molecule NrCAM exists in a variety of isoforms as a result of alternative splicing of individual exons during RNA processing. In this report we demonstrate that many of the alternative splicing events described for chick are conserved in man and describe a novel variant of NrCAM cDNA. Furthermore, we show that NrCAM is expressed at significant levels outside the nervous system; in particular in pancreas, adrenal glands, and placenta and that expression in both brain and other tissues is accompanied by a very variable pattern of exon utilization in fetal and adult cells.

Increased axon growth through astrocyte cell lines transfected with urokinase.

The ability of cells to migrate through tissues depends on their production of a variety of proteases, and the same may be true of growth cones. Urokinase (plasminogen activator) regulates much of the extracellular proteolytic activity, by activating other proteases and as a result of its own proteolytic activity. In order to evaluate the potential role of urokinase as a promoter of axon growth, we have used a plasmid expressing urokinase under a cytomegalovirus promoter to transfect an astrocyte cell line, Neu7, which we have previously shown to provide a poor environment for axon regeneration. Five transfected lines all showed greatly increased ability to promote axon regeneration in both monolayer and three-dimensional cultures. The critical change in the transfected cells was largely within the extracellular matrix, since extracellular matrix laid down by urokinase-secreting cells was more permissive to axon growth than matrix from the parent Neu7 line. The effect was due to urokinase since treatment of the transfected cells with the urokinase inhibitors B623 and B428 rendered both the cells and their matrix much less permissive to axon growth, but did not require plasminogen, since it was blocked neither by serum-free medium nor by plasmin inhibitors.

Somatic and germ line mosaicism and mutation origin for a mutation in the L1 gene in a family with X-linked hydrocephalus.

X-linked hydrocephalus is caused by mutations in the gene for neural cell adhesion molecule L1 (L1CAM). In this report, we describe identification of a mutation in an isolated case of hydrocephalus with adducted thumbs. Tracing the origin of the mutation within the family showed a degree of somatic mosaicism in the asymptomatic maternal grandfather of the propositus. This report highlights the need to take mosaicism into account when counselling relatives of affected individuals.

Nine novel L1 CAM mutations in families with X-linked hydrocephalus.

Mutations in the gene for neural cell adhesion molecule L1 are responsible for the highly variable phenotype found in families with X-linked hydrocephalus, MASA syndrome, and spastic paraplegia type I. To date, 32 different mutations have been observed, the majority being unique to individual families. Here, we report nine novel mutations in L1 in 10 X-linked hydrocephalus families. Four mutations truncate the L1 protein and eliminate cell surface expression, and two would produce abnormal L1 through alteration of RNA processing. A further two of these mutations are small in-frame deletions that have occurred through a mechanism involving tandem repeated sequences. Together with a single missense mutation, these latter examples contribute to the growing number of existing mutations that affect short regions of the L1 protein that may have particular functional significance.

Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders.

The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties.

Bridge grafts of fibroblast growth factor-4-secreting schwannoma cells promote functional axonal regeneration in the nigrostriatal pathway of the adult rat.

Axons damaged in the adult mammalian central nervous system are able to regenerate when their inhibitory glial environment is replaced with a more permissive substrate. Here, we have used long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells to allow mechanically lesioned nigrostriatal axons to regenerate back to their original target in the adult rat brain. Regenerated axons were able to leave the bridge graft to form terminal arborizations and increase the density of tyrosine hydroxylase-immunoreactive fibres within the striatum. Bridge grafting also resulted in an increase in the number of neurons within the substantia nigra pars compacta taking up the fluorescent retrograde tracer Fluoro-Gold from the striatum. Animals which had received RN-22 bridge grafts showed lower rates of amphetamine-induced rotation 10 weeks after a mechanical lesion of the nigrostriatal tract compared to lesioned controls, the magnitude of the behavioural effect being related to the number of regenerated axons, and this comparative reduction was reversed by mechanical section of the bridge graft. It is concluded that our bridge grafting strategy allowed the partial anatomical and functional regeneration of the mechanically lesioned nigrostriatal tract, an unmyelinated central axon bundle, and that bridge grafting therefore represents a realistic approach to the repair of central nervous system lesions involving axon tract damage.

Functional and anatomical reconstruction of the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat.

In an attempt to reconstruct the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat we have combined homotopic grafting of embryonic ventral mesencephalon suspensions with the implantation of long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells stretching from the site of the neuronal grafts to the striatum. At seven weeks after receiving both grafts, animals were killed and processed for immunohistochemistry against tyrosine hydroxylase. Tyrosine hydroxylase-immunoreactive axons were seen to extend from the nigral grafts, along the bridge graft to the striatum where terminal arborizations could be seen. The retrograde tracer Fluoro-gold was injected intrastriatally in some of the experimental animals and was taken up by grafted neurons confirming their projection to the striatum. In parallel to the anatomical reconstruction of the system, a decrease in amphetamine-induced rotation was demonstrated in those animals receiving both grafts which had received > 98% complete lesions. This decrease was greatest in those animals with the most tyrosine hydroxylase-immunoreactive axons in their bridge grafts. The presence of the bridge graft also led to an increase in neuronal graft survival with twice as many tyrosine hydroxylase-immunoreactive neurons being found in the grafts of those animals that had received both grafts compared to those that had received a neuronal graft but no bridge graft.

In vivo effects of kFGF on embryonic nigral grafts in a rat model of Parkinson's disease.

Central injections of FGF have been reported to promote the survival of dopamine neurones in nigral grafts. With the goal of developing an improved delivery of trophic molecules, an immortalized RN22 Schwann cell line transfected with a secretory form of FGF, kFGF, was irradiated and co-transplanted with embryonic nigral grafts in the 6-OHDA lesioned rat striatum. Amphetamine-induced turning was alleviated by nigral grafts, but was not further improved by co-grafts, whether or not transfected to secrete kFGF. Histological analysis showed similar numbers of surviving transplanted cells and a similar extent of fibre growth from the nigral grafts whether implanted alone or co-grafted with the Schwann cells. These results suggest that kFGF does not have any clear in vivo effect on embryonic nigral grafts in this model.

An analysis of astrocytic cell lines with different abilities to promote axon growth.

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.

Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene.

The expression of genes encoding patatin, a major tuber protein, is highly tissue-specific but is also modulated by exogenous sucrose. The patterns of transcription observed in potato plants could be due to mechanisms conferring tuber-specificity or they could reflect the concentrations of sucrose found in different tissues. To distinguish between these possibilities, a detailed examination was made of the function of a region of the promoter previously implicated in conferring tissue-specific and sucrose-inducible expression. Internal deletions of this region revealed three separate functional domains regulating expression. The B repeat region acted as a positive activator of transcription in the tuber and was also responsible for a degree of sucrose-inducibility. The distal region of the A repeat repressed transcription in leaf and tuber tissue, while the proximal region of the A repeat was able to confer sucrose-responsiveness. Each of these regions specifically bound nuclear proteins which may be putative transcription factors involved in conferring these responses. The region found to confer sucrose-inducible expression was conserved among some other genes that are also regulated by exogenous sucrose.

An inhibitor of neurite outgrowth produced by astrocytes.

We have produced a number of astrocytic cell lines, some of which promote abundant neurite outgrowth, some of which are poor promoters of neurite outgrowth. The critical difference between these lines lies in the extracellular matrix, cell lines that are good promoters of axon growth producing a matrix that promotes axon growth, cell lines that are poor promoters of axon growth producing a non-permissive matrix. We were unable to find any consistent correlations between promotion of axon growth and production of proteases, protease inhibitors, N-cadherin, growth cone collapsing activity, and several extracellular matrix molecules. In the present study we have compared the least permissive of our cell lines, Neu7, with the most permissive, A7. Medium conditioned by the cell lines has the same properties as the matrix, since dorsal root ganglia (DRGs) grown in conditioned medium from the Neu7 line grow axons poorly, while DRGs grown in medium conditioned by A7 or primary astrocytes grow many long axons. Since matrix produced by all the cell lines contains large amounts of laminin, we looked to see whether the cells were producing laminin-blocking activity. Medium from the Neu7 line blocked laminin, while that from the A7 and primary astrocytes did not. However, when the conditioned media were heat-treated to remove neurite-promoting activity, they all had laminin-blocking activity: the blocking activity is heat stable. The neurite-promoting properties of the conditioned media therefore probably reflect a balance between promoting molecules and blockers.(ABSTRACT TRUNCATED AT 250 WORDS)

The changes of circular dichroism and fluorescence spectra, and the comparison with inactivation rates of angiotensin converting enzyme in guanidine solutions.

The circular dichroism (CD) spectrum of angiotensin converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) in the ultraviolet region was shown to have down negative peaks at 208 and 222 nm, indicating its peptide chain has an alpha-helical structure. The conformational changes of the enzyme during denaturation in guanidine solutions of increasing concentration, for 24 h at 4 degrees C, were associated with the disappearance of the two negative peaks of the CD spectra, less alpha-helical structure to various extents, a decrease in intensity of the intrinsic protein fluorescence, a red shift in the emission maximum at 340 nm and an increase in the band-width of the spectrum delta lambda. Together these findings demonstrate unfolding of the folded peptide chain of angiotensin converting enzyme and consequent exposure of its aromatic amino acid residues during denaturation. The rates of ellipticity (theta 220) changes of the enzyme during denaturation were less than those of the decrease in fluorescence intensity, demonstrating that the rate of degradation of its secondary structure was slower than that of its tertiary structure. Both the rates of inactivation and conformational change of the enzyme increased with increasing guanidine concentrations, within the range of 1.0-3.0 M. The enzyme inactivation had separate fast and slow processes. Both the rates and the extents of inactivation were much faster and larger than those of conformational changes. Compared with other enzymes, therefore, the angiotensin converting enzyme molecule appears to have a stable spatial structure, but its active site conformation is relatively unstable during denaturation.