Diffusion tractography of the fornix in schizophrenia.

BACKGROUND: White matter fiber tracts, especially those interconnecting the frontal and temporal lobes, are likely implicated in pathophysiology of schizophrenia. Very few studies, however, have focused on the fornix, a compact bundle of white matter fibers, projecting from the hippocampus to the septum, anterior nucleus of the thalamus and the mamillary bodies. Diffusion Tensor Imaging (DTI), and a new post-processing method, fiber tractography, provides a unique opportunity to visualize and to quantify entire trajectories of fiber bundles, such as the fornix, in vivo. We applied these techniques to quantify fornix diffusion anisotropy in schizophrenia. METHODS: DTI images were used to evaluate the left and the right fornix in 36 male patients diagnosed with chronic schizophrenia and 35 male healthy individuals, group matched on age, parental socioeconomic status, and handedness. Regions of interest were drawn manually, blind to group membership, to guide tractography, and fractional anisotropy (FA), a measure of fiber integrity, was calculated and averaged over the entire tract for each subject. The Doors and People test (DPT) was used to evaluate visual and verbal memory, combined recall and combined recognition. RESULTS: Analysis of variance was performed and findings demonstrated a difference between patients with schizophrenia and controls for fornix FA (p=0.006). Protected post-hoc independent sample t-tests demonstrated a bilateral FA decrease in schizophrenia, compared with control subjects (left side: p=0.048; right side p=0.006). Higher fornix FA was statistically significantly correlated with DPT and measures of combined visual memory (r=0.554, p=0.026), combined verbal memory (r=0.647, p=0.007), combined recall (r=0.516, p=0.041), and combined recognition (r=0.710, p=0.002) for the control group. No such statistically significant correlations were found in the patient group. CONCLUSIONS: Our findings show the utility of applying DTI and tractography to study white matter fiber tracts in vivo in schizophrenia. Specifically, we observed a bilateral disruption in fornix integrity in schizophrenia, thus broadening our understanding of the pathophysiology of this disease.

Neutral protease inhibitors from human intervertebral disc and femoral head articular cartilage.

Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocytes containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus. It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus.

Evidence supporting a role for cathepsin B in the generation of T cell antigenic epitopes of human growth hormone.

The elucidation of the enzymatic processing mechanism associated with the formation of antigenic peptide fragments that combine with MHC class II molecules is fundamental to our understanding of the immune system. We have investigated a structurally well defined protein, recombinant human growth hormone (rhGH), as an antigen, and present data supporting the hypothesis that the enzyme cathepsin B can produce peptide fragments bearing T cell epitopes associated with lymphocyte proliferative response to hGH in Balb/c (H-2dhaplotype) mice. Minimal T cell epitopes are not generated; rather the cathepsin cleavage sites flank the three antigenic peptide regions, amino acid residues 31-41, 81-100, and 166-181.

An antigenic complex in the rhoptries of Plasmodium falciparum.

A previously identified putative rhoptry antigen of Plasmodium falciparum is composed of two major components, one of 80 kDa and a doublet at 42/40 kDa. An inhibitory monoclonal antibody immunoprecipitated both the 80 kDa protein and the 42/40 kDa doublet, but immunoblotted only the 80 kDa component. A second monoclonal antibody, raised against the affinity purified complex, immunoblotted only the 42 kDa band under non-reducing conditions. Electron microscopic examination of thin sections of parasites immunolabeled with these monoclonal antibodies and colloidal gold anti-mouse conjugate has confirmed that this antigen is localised in the rhoptry organelles of mature schizonts and free merozoites. The antigen is associated with apparent membranous structures released from free merozoites. Immunoblotting and immunoprecipitation with two different monoclonal antibodies, and protease digestion experiments, have clearly demonstrated that this antigen is a complex composed of two separate and distinct proteins, and does not represent a monomer/dimer pair. The 80 kDa protein is synthesised as an 84 kDa precursor.

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies.

Four monoclonal antibodies produced against Plasmodium falciparum recognize an antigen in merozoites that is localized in rhoptries, as judged by a punctate, double dot fluorescence pattern. All four antibodies bound to the same affinity purified antigen in a two site immunoradiometric assay. Immunoprecipitation of antigen by monoclonal antibody followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis yielded protein bands of 80, 66 and 42 kDa. Western blotting gave bands of 80 and 66 kDa only with three of the antibodies: the fourth did not blot. Based on protease inhibitor data the 66 kDa band is considered to be a cleavage product of the 80 kDa band, but the 42 kDa band does not appear to derive from the latter and may be a coprecipitation product. This group of antigens labels with both [35S]methionine and [3H]histidine. Two of the monoclonal antibodies inhibited merozoite invasion of erythrocytes. One of these inhibitors recognizes a variable epitope, whereas the second recognizes a highly conserved epitope present in all 106 primary isolates of P. falciparum tested from Brazil, Thailand and Papua New Guinea.

An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum.

Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. These antibodies recognise a glycosylated antigen of approximately 51 kDa which is located on the merozoite surface membrane.

Cloning and characterisation of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains.

A dextran sulphate protein (DSP) fraction derived from Babesia bovis has previously been shown to induce a protective immune response in cattle. A B. bovis cDNA library was screened with both the complete anti-DSP serum and a subfraction of the anti-DSP serum affinity purified on a native B. bovis protein of approx. 80 kDa. cDNA clones encoding two different B. bovis proteins were identified. The product of one gene, Bv80, has a single divergent copy of a sequence of 149 amino acids (approx. 30% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are separated by an array of short variant repeat sequences rich in proline and glutamic acid. The product of the other gene, BvVAl (homologous to the previously described 225-kDa B. bovis protein)[19], is predicted to have a single divergent copy of a sequence of 170-171 amino acids (approx. 35% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are also separated by an array of repeats. The 73-amino acid repeat unit of this array is composed of a number of variant derivatives of shorter repeat units. Detailed analysis of genomic clones flanking two alleles of the gene encoding BvVAl/225 kDa identified further members of a multi-gene family. This region of the genome of B. bovis has been subject to a large number of amplification processes.

Isolation and partial characterisation of a 26 kilodalton antigen from Plasmodium falciparum recognised by an inhibitory monoclonal antibody.

A 26 kDa protein, present in trophozoites and schizonts of Plasmodium falciparum, has been identified as the target of a monoclonal antibody that weakly inhibits parasite growth in vitro. The antigen has been purified to homogeneity by immuno-affinity chromatography and electrophoresis. The sequence of 19 amino acids at the N-terminus of the protein has been determined.

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides.

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.

Jun, Fos and Krox in the thalamus after C-fiber stimulation: coincident-input-dependent expression, expression across somatotopic boundaries, and nucleolar translocation.

Expression of the inducible transcription factors Jun, Fos and Krox is commonly used to map neurons in the brain that are activated by sensory inputs. However, some neurons known to be electrically excited by such inputs do not always express these factors. In particular, stimulation of hindlimb sensory nerve C-fibers induces expression of c-Fos in the medial thalamus (the mediodorsal, intermediodorsal, centrolateral and centromedial), but not in the lateral thalamus (the ventroposterolateral, ventroposteromedial and posterior group). We hypothesized that c-Fos expression might only occur in these lateral areas after more complex stimulation patterns, or that only other transcription factors can be induced in these areas by such stimuli. Thus we examined the effects of single, repeated and coincident C-fiber inputs on expression of six inducible transcription factors in the medial, lateral and reticular thalamus of the rat. A weak C-fiber input caused by noxious mechanical stimulation of the skin of one hindpaw did not induce expression of c-Fos, FosB, Krox-20 or Krox-24; but it did reduce the basal expressions of c-Jun and JunD in both the medial and lateral areas. An intense input produced by electrical stimulation of all the C-fibers in one sciatic nerve also failed to induce expression of c-Fos, FosB, Krox-20 or Krox-24 in the medial or lateral areas. However, in the medial thalamus it increased c-Jun and reduced the basal expression of JunD, whereas in the lateral thalamus it had no effect on c-Jun but again reduced the basal expression of JunD. With repeated stimulation, i.e. when the noxious stimulus was applied to the contralateral hindpaw 6 h after the sciatic stimulation, there was again no induction of c-Fos, FosB or Krox-20 in the medial thalamus; but there was an increase in c-Jun and Krox-24, and a decrease in JunD levels. In the lateral thalamus the repeated stimulation again failed to induce c-Fos, but the expressions of FosB, c-Jun and Krox-24 were increased, and that of JunD was again reduced. With coincident stimulation, i.e. when a stimulus was applied to each hindpaw simultaneously, c-Fos and Krox-24 remained absent; but there was a marked induction of FosB and Krox-20, a strong repression of c-Jun, and no effect or a reduction of the basal levels of JunD. This coincident stimulation also caused FosB to appear in the nucleolus of many thalamic neurons. MK-801, but not L-NAME, blocked all these changes. In summary, noxious stimulation affects the expression of all transcription factors in the medial, lateral and reticular thalamus in a complex manner depending upon the inducible transcription factor considered, the thalamic nucleus, and the stimulation paradigm. The expression of some transcription factors uniquely after simultaneous inputs suggests they act as coincidence detectors at the gene level.

Concentration of Babesia bigemina-infected erythrocytes using a dextran sulphate affinity column.

Babesia bigemina-infected erythrocytes preferentially bind to dextran sulphate affinity columns. Subsequent elution yields suspensions containing up to 95% infected erythrocytes. Preliminary immunoblotting studies indicate a parasite antigen of 35,000 mol. wt might be implicated in the binding.

Vaccination of cattle with dextran sulphate-binding Babesia bigemina antigens.

Dextran sulphate-bound Babesia bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphate-binding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions--one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemina-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group.

Babesia bovis host cell recognition proteins.

Babesia bovis enters host erythrocytes by invagination but nothing is known of the proteins involved. By means of metabolic labelling, differential centrifugation in oil and salt elution, a number of babesial proteins have been shown to bind to bovine erythrocytes. Strong binding is evidenced only by a 38/19 kDa pair. Preliminary experiments indicate that these two proteins also bind to human erythrocytes, although apparently to a lesser extent.

Differentiation in an olfactory cell line. Analysis via differential display.

The olfactory epithelium is a unique system, in which new neurons are continually generated throughout adult life. Olfactory neurons are derived from stem cells that lie adjacent to the basal lamina of the olfactory epithelium; these stem cells divide several times and their progeny differentiate into mature sensory neurons. In our laboratory immortalized cell lines have been derived from these dividing cells. The morphology of these cell lines and their expression of neuronal markers varies with culture conditions. When grown in low serum medium one of these cells lines, OLF 442, differentiates by extending long neurites and increasing its expression of neurofilament and B50/GAP43 proteins at the same time reducing expression of glial fibrillary acidic protein (GFAP). Identification of differentially expressed mRNA in cell lines has previously relied on both screening for known markers, and the use of subtractive techniques for identification of unique mRNA species. The differential display technique allows simultaneous detection of differentially expressed mRNA at different time periods and growth conditions. A modified Liang and Pardee differential display technique was used to screen OLF 442 over a number of time intervals in serum-depleted media, and compared with OLF 442 grown in complete media. The differentially displayed fragments were cloned and sequenced, leading to the identification of a number of sequences, both known and unknown. The known sequences include SPARC (encoding a Ca2+ binding secreted Protein which is Acidic and Rich in Cysteine), which is reported to function as a modulator of the cell matrix, and RHAMM, the receptor for hyaluronan-mediated motility. Both the known and the unknown sequences are being studied further to provide insight into the differentiation of olfactory neurons.

Neurogenesis in adult human.

This report describes neurogenesis in the adult human olfactory epithelium in vitro. Olfactory epithelium was collected at autopsy and by biopsy, and grown in serum-free medium. Basic fibroblast growth factor induced the differentiation of bipolar cells which were immunopositive for several neuronal proteins but not glial proteins. [3H]thymidine autoradiography confirmed that these neurones were born in vitro. The results demonstrate that the adult human olfactory epithelium retains the capacity for neurogenesis and neuronal differentiation, at least until the age of 72 years. It is now possible to examine neurones and neurogenesis in biopsies from patients with disorders that may involve a neurodevelopmental or neurodegenerative aetiology such as schizophrenia, bipolar disorder and Alzheimer's disease.

On techniques for the measurement of the mass fractal dimension of aggregates.

A review is presented of a number of techniques available for the characterisation of the structure of aggregates formed from suspensions of sub-micron particles. Amongst the experimental techniques that have been commonly used are scattering (light, X-ray or neutron), settling and imaging and these are the focus of this work. The theoretical basis for the application of fractal geometry to characterisation of flocs and aggregates is followed by a discussion of the strengths and limitations of the above techniques. Of the scattering techniques available, light scattering provides the greatest potential for use as a tool for structure characterisation even though interpretation of the scattered intensity pattern is complicated by the strong interaction of light and matter. Restructuring further complicates the analysis. Although settling has long been used to characterise particle behaviour, the absence of an accurate permeability model limits the technique as a means of determining the porosity of fractal aggregates. However, it can be argued that the determination of fractal dimension is relatively unaffected. The strength of image analysis lies in its ability to provide a great deal of information about particle morphology and the weaknesses lie in the difficulties with image processing and sample size as this is a particle counting technique. There are very few papers which compare the fractal dimension measured by more than one technique. Light scattering potentially provides a useful tool for checking settling results. However, further work is required to develop proper models for aggregate permeability and flow-through effects.

Distribution of glycosaminoglycans across the normal and the scoliotic disc.

This paper reports on our investigations of the glycosaminoglycans (GAGs) (hyaluronic acid, chondroitin-6-sulfate, and keratan sulfate) of 13 adjacent zones of normal and scoliotic discs obtained at necropsy. The isolation and separation of these GAGs on the microgram scale was achieved using the different solubilities of the calcium and cetyl pyridinium chloride complexes. In normal discs the distribution of these GAGs is symmetric about the nucleus pulposus where their concentration is highest, and lowest in the outer annulus regions. A shift in this distribution profile was observed for the GAGs of the scoliotic discs, particularly at the apex of the curve. On the concave side of this disc the chondroitin-6-sulfate was elevated relative to discs at a lower level. In those annulus zones on the convex side of the curve, the keratan sulfate and hyaluronic acid levels were elevated and chondroitin sulfate depressed. These results suggest the presence in these respective regions of at least two different species of proteoglycans accompanied by elevated hyaluronate levels.