Efficacy of vaccination with a Klebsiella pneumoniae siderophore receptor protein vaccine for reduction of Klebsiella mastitis in lactating cattle.

Clinical mastitis caused by Klebsiella spp. is an emerging problem in the US dairy industry and results in a high degree of financial losses to dairy workers. This study was conducted as a randomized, blinded, and placebo-controlled efficacy study of a Klebsiella pneumoniae siderophore receptor protein (SRP) vaccine (Kleb-SRP), with a total of 569 cows and heifers enrolled. The study was designed to look at vaccine effect on Klebsiella mastitis; however, the SRP in Klebsiella are highly conserved across coliform bacteria, which means that the vaccine has potential for cross-protection against all coliforms. Cows were paired based on parity, days in milk at enrollment, and somatic cell count. Within pairs, individuals were randomized to receive either Kleb-SRP or a placebo formulation. Following vaccination, the incidence of Klebsiella spp. and total coliform mastitis from natural exposure were compared to determine the efficacy of the vaccine. When analyzing all cows, the reduction of mastitis risk was not significant, though milk production increased 0.31 kg/d and somatic cell counts were reduced by 20.1%. When administered before calving, the vaccine reduced the risk of Klebsiella and total coliform mastitis by 76.9 and 47.5% respectively; however, we observed no significant effect when administered after calving. The vaccine, when administered before calving, also increased milk production by an average of 1.74 kg/d and reduced somatic cell counts by 64.8%. When administered after calving, we noted a slight decrease in daily milk production (0.39 kg) but no significant effect on somatic cell counts. All cows in the study (including vaccinates and placebo) received multiple doses of a commercially available licensed Escherichia coli bacterin. It should be noted that this herd was chosen because of the high number of clinical Klebsiella clinical mastitis cases this herd experienced before the trial and the extreme environmental challenge that was present from bedding with dried manure solids. The data from this study demonstrate efficacy of the Kleb-SRP vaccine against Klebsiella mastitis alone and coliform mastitis in general (including all coliforms) when administered before the initiation of a lactation cycle.

Comparison of gait and pathology outcomes of three meniscal procedures for induction of knee osteoarthritis in sheep.

OBJECTIVE(S): Meniscectomy (MX) of sheep induces a well-established animal model of human osteoarthritis (OA). This study compared the clinical (lameness) and pathological outcomes of unilateral, complete medial MX vs two less traumatic and more easily performed meniscal destabilisation procedures. METHODS: Four-year old wethers (n = 6/group) underwent sham operation, cranial pole release (CPR), mid-body transection (MBT) or total MX of the medial meniscus. Joints were assessed for gross pathology (cartilage erosion and osteophytes), histomorphometry, two histopathology scoring methods (modified Mankin-type and Pritzker score), and immunohistology for ADAMTS- and MMP-cleaved neoepitopes, at 12 weeks post-op. Ground reaction forces (GRFs) were determined by force plate in a subset (n = 4/group) at baseline, 2.5, 8, and 12 weeks post-op. RESULTS: Gross pathology scores of operated groups differed significantly from sham animals (P < 0.05) but not from each other, though qualitative differences were noted: CPR sheep developed more cranial and focal lesions, while MBT and MX joints showed more widespread lesions and osteophyte formation. Similarly, histopathology scores were significantly elevated vs sham but did not differ between operated groups at P < 0.05, except for a trend for lower tibial cartilage histopathology in MBT consistent with the immunohistologic pattern of reduced aggrecanase-cleavage neoepitope in that model. CPR sheep developed less femoral subchondral sclerosis, suggesting some residual biomechanical effect from the destabilised but intact meniscus. Few significant differences were noted between operated groups in force plate analyses, though gait abnormalities appeared to be least in CPR sheep, and most persistent (>12 weeks) in MBT animals. CONCLUSION: The well-validated ovine MX model and the simpler meniscal destabilisation procedures resulted in broadly similar joint pathology and lameness. Meniscal CPR or MBT, as easier and more clinically relevant procedures, may represent preferred models for the induction of OA and evaluation of potential disease-modifying therapies.

Escherichia coli O157:H7 shedding in vaccinated beef calves born to cows vaccinated prepartum with Escherichia coli O157:H7 SRP vaccine.

Extensive research, intervention equipment, money, and media coverage have been directed at controlling Escherichia coli O157:H7 in beef cattle. However, much of the focus has been on controlling this pathogen postcolonization. This study was conducted to examine the performance, health, and shedding characteristics of beef calves that were vaccinated with an E. coli O157:H7 SRP bacterial extract. These calves had been born to cows vaccinated prepartum with the same vaccine. Cows and calves were assigned randomly to one of four treatments: (i) neither cows nor calves vaccinated with E. coli O157:H7 SRP (CON), (ii) cows vaccinated with E. coli O157:H7 SRP prepartum but calves not vaccinated (COWVAC), (iii) calves vaccinated with E. coli O157:H7 SRP but born to cows not vaccinated (CALFVAC), (iv) cows vaccinated with E. coli O157:H7 SRP prepartum and calves also vaccinated (BOTH). Calves born to vaccinated cows had significantly higher titers of anti-E. coli O157:H7 SRP antibodies (SRPAb) in circulation at branding time (P < 0.001). Upon entry to the feedlot, overall fecal E. coli O157:H7 prevalence was 23 % among calves, with 25 % in the CON treatment group, 19 % in the CALFVAC group, 32 % in the COWVAC group, and 15 % in the BOTH group (P > 0.05). Fecal shedding of E. coli O157 on arrival to the feedlot was not correlated with fecal shedding at slaughter (Spearman's rho = -0.02; P = 0.91). No significant effects of cow or calf E. coli O157:H7 SRP vaccination treatment were found on feedlot calf health or performance (P > 0.05), prevalence of lung lesions or liver abscess (P > 0.05), or morbidity, retreatment, or mortality numbers (P > 0.05). The findings of this study indicate that the timing of vaccination of calves against E. coli O157:H7 may be an important consideration for maximizing the field efficacy of this vaccine.

Matrix metalloproteinase 13-deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development.

OBJECTIVE: To investigate the role of matrix metalloproteinase 13 (MMP-13; collagenase 3) in osteoarthritis (OA). METHODS: OA was surgically induced in the knees of MMP-13-knockout mice and wild-type mice, and mice were compared. Histologic scoring of femoral and tibial cartilage aggrecan loss (0-3 scale), erosion (0-7 scale), and chondrocyte hypertrophy (0-1 scale), as well as osteophyte size (0-3 scale) and maturity (0-3 scale) was performed. Serial sections were stained for type X collagen and the MMP-generated aggrecan neoepitope DIPEN. RESULTS: Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (P<0.01) and tibial cartilage erosion increased with time (P<0.05) in wild-type mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (P<0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. There was less tibial cartilage erosion in knockout mice than in wild-type mice at 8 weeks (P<0.02). Cartilaginous osteophytes were larger in knockout mice at 4 weeks (P<0.01), but by 8 weeks osteophyte maturity and size were no different from those in wild-type mice. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both wild-type and knockout mouse joints. CONCLUSION: Our findings indicate that structural cartilage damage in a mouse model of OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.

Effects of a siderophore receptor and porin proteins-based vaccination on fecal shedding of Escherichia coli O157:H7 in experimentally inoculated cattle.

The efficacy of a vaccine containing outer membrane siderophore receptor and porin (SRP) proteins for reducing fecal prevalence and shedding of Escherichia coli O157:H7 was evaluated in cattle inoculated with E. coli O157:H7. Thirty calves were randomly assigned to one of two groups, and on days 1 and 21 these calves were given subcutaneous injections of either a placebo (control) or the vaccine. Blood was collected weekly to monitor the serum anti-SRP antibody titers. Two weeks after the second vaccination, calves were orally inoculated with a mixture of five strains of nalidixic acid-resistant (NalR) E. coli O157:H7. Fecal samples and rectoanal mucosal swabs were collected daily for the first 5 days and then three times each week for the following 4 weeks to determine the presence and enumerate the fecal concentration of NalR E. coli O157:H7. At necropsy on day 35, gut contents and tissue swabs were collected to determine the presence and concentration of NalR E. coli O157:H7. Vaccinated cattle had significantly higher anti-SRP antibody titers than did control cattle, with a significant treatment x week interaction (P < 0.01). Vaccination of cattle with the SRP protein tended to decrease fecal concentration (1.9 versus 1.6 log CFU/g) of NalR E. coli O157:H7 (P = 0.10). The number of calves that were fecal culture positive for E. coli O157:H7 was lower (P = 0.05) in the vaccinated group than in the control group. The E. coli O157:H7 SRP vaccine tended to reduce fecal prevalence and concentration of E. coli O157:H7 in cattle orally inoculated with NalR E. coli 0157:H7 and may be a useful prehavest intervention strategy. Future research must be conducted on natural prevalence in feedlot operations to further evaluate the efficacy of this novel vaccine.

Calcification in the ovine intervertebral disc: a model of hydroxyapatite deposition disease.

The study design included a multidisciplinary examination of the mineral phase of ovine intervertebral disc calcifications. The objective of the study was to investigate the mineral phase and its mechanisms of formation/association with degeneration in a naturally occurring animal model of disc calcification. The aetiology of dystrophic disc calcification in adult humans is unknown, but occurs as a well-described clinical disorder with hydroxyapatite as the single mineral phase. Comparable but age-related pathology in the sheep could serve as a model for the human disorder. Lumbar intervertebral discs (n = 134) of adult sheep of age 6 years (n = 4), 8 years (n = 12) and 11 years (n = 2) were evaluated using radiography, morphology, scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray powder diffraction, histology, immunohistology and proteoglycan analysis. Half of the 6-year, 84% of the 8-year and 86% of the 11-year-old discs had calcific deposits. These were not well delineated by plain radiography. They were either: (a) punctate deposits in the outer annulus, (b) diffuse deposits in the transitional zone or inner annulus fibrosus with occasional deposits in the nucleus, or (c) large deposits in the transitional zone extending variably into the nucleus. Their maximal incidence was in the lower lumbar discs (L4/5-L6/7) with no calcification seen in the lumbosacral or lower thoracic discs. All deposits were hydroxyapatite with large crystallite sizes (800-1,300 A) compared to cortical bone (300-600 A). No type X-collagen, osteopontin or osteonectin were detected in calcific deposits, although positive staining for bone sialoprotein was evident. Calcified discs had less proteoglycan of smaller hydrodynamic size than non-calcified discs. Disc calcification in ageing sheep is due to hydroxyapatite deposition. The variable, but large, crystal size and lack of protein markers indicate that this does not occur by an endochondral ossification-like process. The decrease in disc proteoglycan content and size suggests that calcification may precede or predispose to disc degeneration in ageing sheep.

Evidence for articular cartilage regeneration in MRL/MpJ mice.

OBJECTIVE: A major clinical problem in Orthopaedics is the repair of traumatic articular cartilage lesions. The MRL/MpJ strain of mice has the remarkable ability to regenerate ear hole punch wounds seamlessly including the scarless replacement of multiple tissues. The objective of this study was to assess whether articular cartilage defects repair or regenerate in the MRL/MpJ 'healer' strain of mice. METHOD: Full thickness and partial thickness lesions were introduced into trochlear groove articular cartilage of MRL/MpJ and C57Bl/6 mice, a control strain that does not undergo ear hole regeneration. The wound sites were assessed 6 weeks and 12 weeks post-surgery using a histological scoring scheme and immunohistochemistry for markers of articular cartilage including proteoglycan, collagen II and collagen VI. RESULTS: The partial thickness lesions did not repair in either strain. However, at both 6 weeks and 12 weeks timepoints the MRL/MpJ mice had a superior healing response of full thickness lesions with abundant chondrocytes and an extracellular matrix rich in proteoglycan, collagen II and collagen VI at the wound site. At the 12 week timepoint the enhanced cartilage healing was restricted to male MRL/MpJ mice. In contrast, the C57Bl/6 control strain produced an extracellular matrix at the wound site that, overall, had significantly less matrix proteoglycan and collagen II. CONCLUSIONS: Male MRL/MpJ mice appear to possess an intrinsic ability to 'regenerate' articular cartilage. Understanding the biochemical and genetic basis for articular cartilage regeneration may open up new treatment options for traumatic articular cartilage defects.

Transcranial grey-scale sonography of subdural haematoma in adults.

AIM: Transcranial high-resolution grey-scale sonography reliably allows diagnosis and monitoring of subdural haematoma (SDH) and extra-cerebral intracranial fluid collections in infants but has not been evaluated thoroughly in adults up to now. Because of rapid development of ultrasound systems, the depiction of intracerebral haemorrhage (ICH) has now become feasible. The presented study evaluated the sonographic appearance of SDH in adults. METHOD: We performed transcranial grey-scale sonography (TGS) in 25 consecutive patients with SDH confirmed by cranial computed tomography (CCT) or MRI. According to paediatric TGS, the dural border of the arachnoid was depicted as a highly echogenic membrane, and the distance between the skull and the echogenic membrane was measured. SDH was measured by CCT/MRI and by TGS in corresponding axial planes. The rate of identification of SDH in TGS was evaluated, and the extent of SDH as assessed by CCT/MRI and TGS was compared. RESULTS: TGS reliably detected SDH in 22 of the 25 patients with confirmed SDH (88 %). In the remaining 3 patients, the temporal bone window was insufficient for TGS investigation. Extent of SDH measured by CCT and TGS correlated linearly (r= 0.849). CONCLUSION: TGS allows imaging of SDH in patients with CCT/MRI confirmed SDH, and the extent of SDH correlates significantly between TGS and CCT/MRI. Therefore, TGS may be a possible alternative to serial CCT imaging in monitoring SDH, since in contrast to CCT, TGS is a non-invasive bedside method. So far, TGS is not suitable for the diagnosis of SDH.

Topographical analysis of the structural, biochemical and dynamic biomechanical properties of cartilage in an ovine model of osteoarthritis.

OBJECTIVE: The relationship between the topographical variations in the structural, biochemical and dynamic biomechanical properties of articular cartilage (AC) before and 6 months after meniscectomy has not been previously reported but is clearly relevant to our understanding of the role of mechanical factors on the pathogenesis of osteoarthritis (OA). The objective of this study was to address this deficiency using an ovine model of OA induced by bilateral lateral meniscectomy. DESIGN: The dynamic effective shear modulus (G*) and phase lag were determined ex vivo at 26 individual locations over the medial and lateral tibial plateaux of non-operated and meniscectomized ovine joints 6 months after surgery using a novel hand-held dynamic indentation probe. AC thickness was measured with a needle penetration probe. The AC from the same topographical locations as indented was then analysed for sulfated glycosaminoglycans (S-GAG) as a measure of proteoglycan (PG) levels, collagen and water content. Histological evaluation of the collagen organization using quantitative analysis of birefringence intensity was performed on stained tissue sections from the same topographical locations of each animal. RESULTS: It was demonstrated that the AC of the entire lateral tibial compartment of the meniscectomized joints underwent significant local degenerative and compensatory changes as indicated by a decreased G* and an increase in phase lag and water content. This was accompanied by a decrease in PG content of the AC of the middle and inner regions. While the AC of the outer region of the lateral meniscectomized compartment showed a marked increase in PG content and a more than two-fold increase in thickness, these tissues were also found to be structurally inferior, as indicated by a decreased G* and abnormal collagen birefringence intensity. The AC thickness was elevated at all locations of the lateral and medial tibial plateau of the meniscectomized joints. Strong and significant correlations between the biomechanical and biochemical data were established for a number of the parameters examined, especially between collagen content and G*, collagen content and AC thickness, and G* and AC thickness. An inverse correlation between S-GAG content and G* was only apparent in non-operated control tissues, whereas correlations between collagen and water content, water content and G*, and water content and thickness were evident for AC of the meniscectomized tibial plateaux. Less striking changes were noted in the medial compartment where the intact meniscus remained in place. However, elevated PG content, thicker AC together with slight changes in G* suggested an early hypertrophic response in these tissues. CONCLUSION: This study has highlighted the variable response of AC in different topographical regions of meniscectomized joints to the altered mechanical stresses introduced by this surgical procedure. The AC at the joint margins, while thicker and richer in PG, was found to be biomechanically softer (lower shear modulus) than normal AC, and because of this, would be expected to undergo degenerative changes with time leading to the onset of OA.

Decremental intravenous pulse propagation during extrastimulus pacing: relevance for catheter ablation of focal atrial fibrillation.

We report a case study demonstrating delayed circumferential intrapulmonary-venous conduction characteristics during coronary sinus extrastimulus pacing. This phenomenon allowed the unmasking and discrimination of a localized left atrial to PV breakthrough from secondarily activated PV muscle in a common left-sided PV ostium. Thus, this pacing manoeuvre may serve to guide RF delivery in the treatment of focal AF.

The effects of orally administered diacerein on cartilage and subchondral bone in an ovine model of osteoarthritis.

OBJECTIVE: An ovine model of osteoarthritis (OA) induced by bilateral lateral meniscectomy (BLM) was used to evaluate in vivo effects of the slow acting antiarthritic drug diacerein (DIA) on degenerative changes in cartilage and subchondral bone of the operated joints. METHODS: Twenty of 30 adult age matched Merino wethers were subjected to BLM in the knee joints and the remainder served as non-operated controls (NOC). Half of the BLM group (n = 10) were given DIA (25 mg/kg orally) daily for 3 mo, then 50 mg/kg daily for a further 6 mo. The remainder of the meniscectomized (MEN) group served as OA controls. Five DIA, 5 MEN, and 5 NOC animals were sacrificed at 3 mo and the remainder at 9 mo postsurgery. One knee joint of each animal was used for bone mineral density (BMD) studies. Osteochondral slabs from the lateral femoral condyle and lateral tibial plateau were cut from the contralateral joint and were processed for histological and histomorphometric examination to assess the cartilage and subchondral bone changes. RESULTS: No significant difference was observed in the modified Mankin scores for cartilage from the DIA and MEN groups at 3 or 9 mo. However, in animals treated with DIA, the thickness of cartilage (p = 0.05) and subchondral bone (p = 0.05) in the lesion (middle) zone of the lateral tibial plateau were decreased relative to the corresponding zone of the MEN group at 3 mo (p = 0.05). At 9 mo subchondral bone thickness in this zone remained the same as NOC but BMD, which included both subchondral and trabecular bone, was significantly increased relative to the NOC group (p = 0.01). In contrast, the subchondral bone thickness of the outer zone of lateral tibial plateau and lateral femoral condyle of both MEN and DIA groups increased after 9 mo, while BMD remained the same as in the NOC. CONCLUSION: DIA treatment of meniscectomized animals mediated selective responses of cartilage and subchondral bone to the altered mechanical stresses induced across the joints by this procedure. While subchondral bone thickness in tibial lesion sites was reduced, cartilage and bone proliferation at the outer joint margins, a region where osteophyte formation occurred, were enhanced, suggesting that DIA supported the processes of repair and endochondral ossification.

A novel microassay for the quantitation of the sulfated glycosaminoglycan content of histological sections: its application to determine the effects of Diacerhein on cartilage in an ovine model of osteoarthritis.

OBJECTIVE: A new micro-histological method of assessing the sulfated glycosaminoglycan (S-GAG) content in unstained histological sections of articular cartilage was developed and used to study the effects of orally administered Diacerhein (DIA) on joint cartilage in an ovine model of osteoarthritis (OA). METHODS: Twenty adult, age-matched Merino wethers were subjected to bilateral lateral meniscectomy, while 10 served as non-operated controls (NOC groups). Half of the operated sheep (N=10) remained untreated (MEN groups), while the other 10 animals were given DIA (25 mg/kg orally) daily for 3 months, then 50 mg/kg daily for a further 6 months (DIA groups). Five animals each of the DIA, MEN and NOC groups, respectively, were sacrificed at 3 months post-operatively, and the remainder 6 months later. For the present study only one knee joint of each animal was used for histological processing. The tissues studied were from the lateral femoral condyles (LFC) and lateral tibial plateaux (LTP). Each of these joint regions was further subdivided into inner (I), middle (M), and outer (O) zones. Unstained histological sections from these AC regions and zones were then analysed for S-GAG content using the following procedure. Images of each section of 6 microm thickness were acquired using a flatbed scanner and the area determined with an image analysis software program. The sections were then transferred to wells of a microtiter plate, digested with papain and the S-GAG content quantitated using a modification of the 1,9-dimethylmethylene blue dye binding assay. The data was represented as microg S-GAG/mm(3)of each tissue section. These data were also compared with toluidine blue stained sections from the same paraffin blocks. RESULTS: The results obtained showed that the area of histological sections could be very accurately determined by computer assisted image analysis using a 10 mmx10 mm calibration grid. Cartilage sections of areas ranging from 1 mm(2)up to 25 mm(2)were analysed for S-GAG content with this simple technique. There was a linear relationship between section thickness (2-10 microm) and S-GAG content per unit area (R(2)=0.993). Sections of 6 microm thickness were found to be optimal. S-GAG analyses of serial sections from tibial and femoral articular cartilage (I, M and O zones) revealed an average coefficient of variation of 7.0+/-2.3% (range 4.9-10.2%) confirming the accuracy and reproducibility of this assay method. A separate experiment showed that no significant losses of S-GAG occurred during the histological sample processing. The different regions and zones of the knee joint AC in the six experimental groups revealed variable levels of S-GAG which did not necessarily correlate with the histochemical distribution of toluidine blue staining. The major S-GAG changes occurred in the middle (lesion zone) and outer zones (hypertrophic zone) of both the LFC and LTP of the MEN groups. In the lesion (M) zone the S-GAG content was reduced while in the O zone levels were increased at both 3 and 6 months post-surgery. In animals receiving Diacerhein S-GAG levels in the M zone were lower than or equivalent to those of non-drug treated OA or non-operated controls for both joint regions at 3 and 6 months. While the hypertrophic response in the outer zone of the LFC, as assessed by S-GAG content, was enhanced by drug treatment, the cartilage of the outer zones of the LTP was not affected by drug treatment. CONCLUSION: The results of this study have demonstrated that the S-GAG (and therefore proteoglycan [PG]) content in different cartilage zones of OA joints can be readily quantitated by direct biochemical analysis of unstained histological sections. By this means subtle changes in PG distribution in different cartilage zones, which were not evident using traditional histochemical staining methods, could be readily detected.

Effects of light adaptation on contrast processing in bipolar cells in the retina.

Effects of light adaptation on contrast processing in the outer retina were investigated over nearly four decades of background illumination by analyzing the intracellular responses of 111 bipolar cells, 66 horizontal cells, and 22 cone photoreceptors in the superfused eyecup of the tiger salamander (Ambystoma tigrinum). Light adaptation had striking and similar effects on the average contrast responses of the hyperpolarizing (Bh) and depolarizing (Bd) classes of bipolar cells: Over the lower two decades of background illumination, the contrast gain increased 7-fold to reach values as high as 20-30, the dynamic range and the half-maximum contrast decreased by about 60%, the total voltage range increased some 40%, and contrast dominance changed from highly positive to more balanced. At higher levels of background, most aspects of the contrast response stabilized and Weber's Law then held closely. In this background range, the contrast gain of bipolar cells was amplified some 20X relative to that of cones whereas the corresponding amplification in horizontal cells was about 6X. Differences in the growth of contrast gain with the intensity of the background illumination for cones versus bipolar cells suggest that there are at least two adaptation-dependent mechanisms regulating contrast gain. One is evident in the cone photoresponse such that an approximately linear relation holds between the steady-state hyperpolarization and contrast gain. The other arises between the voltage responses of the cones and bipolar cells. It could be presynaptic (modulation of cone transmitter release by horizontal cell feedback or other mechanisms) and/or postsynaptic, that is, intrinsic to bipolar cells. Contrast gain grew with the background intensity by a larger factor in horizontal than in bipolar cells. This provides a basis for the widely held view that light adaptation increases the strength of surround antagonism in bipolar cells. On average, the effects of light adaptation and most quantitative indices of contrast processing were remarkably similar for Bd and Bh cells, implying that both classes of bipolar cells, despite possible differences in underlying mechanisms, are about equally capable of encoding all primary aspects of contrast at all levels of light adaptation.

Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study.

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus

Spinal biomechanics and aging are major determinants of the proteoglycan metabolism of intervertebral disc cells.

STUDY DESIGN: The proteoglycan metabolism of ovine disc nucleus pulposus and anulus fibrosus cells was investigated in relation to age, spinal level, and intrinsic spinal biomechanical properties. OBJECTIVE: To evaluate the hypothesis that with aging loss of proteoglycans from the lumbosacral disc exceeds that from upper lumbar discs because of its proximity to a rigid segment, the sacrum. SUMMARY OF BACKGROUND DATA: The proteoglycan and associated water of the disc decreases with aging. METHODS: Proteoglycans were extracted directly from the disc tissues using 4 M GuHCl and examined by composite agarose polyacrylamide gel electrophoresis. Disc cells were cultured in alginate beads, and their metabolic activity was assessed by 3H-thymidine incorporation into DNA and by bioreduction of a cell proliferation dye. Newly synthesized proteoglycans were radiolabeled with 35S, and their molecular weight distributions and ability to aggregate with hyaluronan were determined by Sephacryl S1000 gel chromatography. Resident proteoglycans extracted from disc tissues with 4 M GuHCl were similarly evaluated. A group of adult animals also were studied biomechanically to evaluate the range of spinal motion (L4/L5 to L7/S1). RESULTS: In contrast to the neonatal proteoglycan samples, the biosynthesis of proteoglycans by nucleus pulposus cells of adult discs increased progressively toward the sacrum. This correlated with increased metabolic activity. Analysis of the resident proteoglycans by composite agarose polyacrylamide gel electrophoresis indicated that although the aggrecan-1 population was present almost exclusively in the neonatal group, it was the aggrecan-2 population that predominated in the adult discs, and it became progressively more heterogeneous with aging and proximity of the disc to the sacrum. CONCLUSIONS: The proteoglycans of the lumbosacral disc of adult animals turned over faster than proteoglycans of adjacent lumbar discs. The reduced proteoglycan content and ability to aggregate, particularly in the nucleus pulposus of lumbosacral discs, indicated that proteoglycan catabolism exceeded the rate of biosynthesis. These events in the lumbosacral disc are thought to be determined mechanically by its proximity to the sacrum.

Contrast rectification and distributed encoding By ON-OFF amacrine cells in the retina.

The encoding of luminance contrast by ON-OFF amacrine cells was investigated by intracellular recording in the retina of the tiger salamander (Ambystoma tigrinum). Contrast flashes of positive and negative polarity were applied at the center of the receptive field while the entire retina was light adapted to a background field of 20 cd/m(2). Many amacrine cells showed remarkably high contrast gain: Up to 20-35% of the maximum response was evoked by a contrast step of only 1%. In the larger signal domain, C50, the contrast required to evoke a response 50% of the maximum, was often remarkably low: 24 of 25 cells had a C50 value of < or =10% for at least one contrast polarity. Across cells and contrast polarity, the dynamic ranges varied from extremely narrow to broad, thereby blanketing the range of reflectances associated with objects in natural environments. Although some cells resembled "contrast rectifiers," by showing similar responses to contrasts of opposite polarity, many did not. Thus for contrast gain and C50, individual cells could show a strong preference for either negative or positive contrast. In the time domain, the preference was strong and unidirectional: for equal contrast steps, the latency of the response to negative contrast was 20-45 ms shorter than that for positive contrast. The present results, when compared with those for bipolar cells, suggest that, on average, amacrine cells add some amplification, particularly for negative contrast, to the high contrast gain already established by bipolar cells. In the time domain, our data reveal a striking transformation from bipolar to amacrine cells in favor of negative contrast. These and further observations have implications for the input and output of amacrine cell circuits. The present finding of substantial differences between cells reveals a potential substrate for distributed encoding of luminance contrast within the ON-OFF amacrine cell population.

Cytogenetic aberrations in myelodysplastic syndrome detected by comparative genomic hybridization and fluorescence in situ hybridization.

Conventional cytogenetics (CC) is proven as a diagnostic and prognostic factor in myelodysplastic syndrome (MDS). However, CC may be hampered by insufficient metaphase preparation and cannot analyze interphase nuclei. These problems are solved by using comparative genomic hybridization (CGH). The CGH was applied to samples from 45 patients with MDS, and the results were compared with CC and fluorescence in situ hybridization (FISH). The CC detected aberrations in 12 of 45 samples, including chromosomes 3 (n = 1), 5 (n = 9), 7 (n = 2),8(n = 1), 18(n = 1),21 (n = 1), X (n = 1), and Y(n = 2). In one patient, loss of B and C group chromosomes and a marker chromosome were seen. The CGH revealed chromosomal imbalances in 18 of 45 samples, including chromosomes 5 (n = 11), 7 (n = 2), 8 (n = 1), 18(n = 1), 20(n = 1), 21 (n = 1), X (n = 1), and Y (n = 2). All unbalanced aberrations found by CC were detected by CGH, too. In two patients, the CGH found additional aberrations and redefined the aberrations of the chromosomes of the B and C group in one sample. The FISH confirmed these aberrations. Additionally performed FISH for chromosomes 5, 7, and 8 gave normal findings in all patients found to be normal in CC and CGH. The CGH and FISH confirmed the results obtained by CC. All three techniques showed changes of chromosomes 5 and 7 as the most frequent aberrations, emphasizing the importance of these chromosomes in the development of MDS. Furthermore, the CC is proven as the basic technique for cytogenetic evaluation of MDS.