[Cardiovascular disease risk in diabetes patients aged 40 years old and above in China].

Objective: To evaluate the incidence of cardiovascular disease (CVD), ischemic and hemorrhagic cardiovascular events among Chinese diabetic patients aged 40 years and above with different CVD risk levels. Methods: This study enrolled participants aged 40 years and above in 15 provinces from a prospective cohort study, the China-PAR project (Prediction for Atherosclerotic Cardiovascular Disease Risk in China). Participants were categorized into two groups according to the presence or absence of diabetes at baseline. Individuals were further classified into low (0-4.9%), moderate (5%-9.9%) and high risk groups (≥10%), based on predicted ten-year CVD risk using the China-PAR equations. Two followed-up surveys were conducted between 2007 and 2015 to identify CVD events, which were defined as nonfatal acute myocardial infarction, or death due to coronary heart disease, or stroke. Ischemic cardiovascular events included nonfatal acute myocardial infarction, or death due to coronary heart disease, or ischemic stroke. Hemorrhagic cardiovascular events included subarachnoid hemorrhage and intracerebral hemorrhage. The incidences of CVD, ischemic and hemorrhagic cardiovascular events were compared in diabetes and non-diabetes population with different CVD risk levels. Results: This study included 89 209 participants aged 40 years and above, the average follow-up period was 8.5 years. The age was (54.8±9.4) years, and 36 794 (41.2%) were men, and 5 730 (6.4%) were diabetic patients. In diabetes patients aged 40 years and above, 53.7% (3 075/5 730) were at high risk of CVD. Age-and sex-adjusted incidence of CVD, ischemic and hemorrhagic cardiovascular events (1 066.93/100 000 person-years, 824.23/100 000 person-years, and 211.56/100 000 person-years) were significantly lower in diabetes patients than those in non-diabetes population with high CVD risk (1 773.73/100 000 person-years, 1 228.18/100 000 person-years, and 446.49/100 000 person-years) (all P<0.001). Among high CVD risk populations, incidence of ischemic events was significantly higher in diabetic patients than in non-diabetes population (1 638.47/100 000 person-years vs. 1 228.18/100 000 person-years, P<0.001), but incidence of hemorrhagic events tended to be lower in diabetic patients than in non-diabetes population (415.70/100 000 person-years vs. 446.49/100 000 person-years, P=0.635). Incidence of ischemic and hemorrhagic events were similar between diabetes patients and non-diabetes population at low or moderate CVD risk groups (all P>0.05). Conclusions: More than half of diabetes patients aged 40 years and above in China have high CVD risk. The incidence of CVD, ischemic and hemorrhagic cardiovascular events are different in diabetic patients with different CVD risk levels.

Accuracy of detecting vertical root fractures in non-root filled teeth using cone beam computed tomography: effect of voxel size and fracture width.

AIM: To investigate simultaneously the effect of voxel size and fracture width on the accuracy of detecting vertical root fractures (VRFs) in non-root filled teeth when using cone beam computed tomography. METHODOLOGY: Fifty-one of 161 extracted human permanent teeth (16 anterior teeth, 132 premolars and 13 mandibular molars) were selected randomly for VRF induction with two fracture widths. All teeth were scanned with four CBCT units at different voxel sizes provided by the units. Three observers classified the presence or absence of VRF using a 5-point scale. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the ROC curve (AUC) were calculated. AUCs amongst voxel sizes and between the fracture widths were compared using the Z test. Intra- and inter-observer agreement was assessed using weighted Cohen kappa. RESULTS: For the NewTom VGi and ProMax 3D Mid CBCT unit, no significant differences were found amongst voxel sizes for the AUCs, irrespective of the fracture width (P > 0.05). There were significant differences between images scanned with voxel size 250 and 160 µm (P = 0.02), and images scanned with voxel size 250 and 80 µm for AUCs in the narrow VRF group for the 3D Accuitomo 170 unit (P = 0.03). For i-CAT FLX, significant differences were found between the voxel protocols of 300 µm and of the other three voxel sizes for AUC, sensitivity and NPV (P < 0.05). Significant differences between the wide and the narrow VRF groups for AUCs were found for 3D Accuitomo 170 (P = 0.01) and ProMax 3D Mid (P < 0.01). CONCLUSIONS: Cone beam computed tomography was accurate for detecting VRF in non-root filled teeth. Fracture width had an effect on the detection of VRF. The effect of the voxel size on the detection of VRF depended on the CBCT unit used.

Are there differences in Hip Biomechanics after hybrid and cementless resurfacing arthroplasty?

There is a paucity of information regarding the clinical performance of the fully cementless metal-on-metal hip resurfacing designs. We compared the biomechanical reconstruction between the two hips of a group of patients treated with a hybrid resurfacing design on one side and a new, fully cementless version of the same resurfacing design on the other side.We retrospectively identified 20 patients with a hybrid hip resurfacing on one side and a fully cementless device on the contralateral side. The cemented femoral components were implanted with a target angle stem to shaft angle of 140° while the cementless femoral components were implanted with the aim to replicate the natural neck to shaft angle.No significant differences were observed post-operatively in femoral offset or leg length despite implantation with a larger metaphyseal stem to femoral shaft angle in the hybrid group. Both hybrid and cementless designs provide similar biomechanical reconstructions.

The reliability and validity of the Unified Classification System of periprosthetic femoral fractures after hip arthroplasty.

The Unified Classification System has expanded and updated the Vancouver Classification System and applied treatment principles to all periprosthetic fractures. This study assessed the reliability and validity of the Unified Classification System for femur fractures after hip arthroplasty. Thirty-eight radiographs were finally evaluated by 6 observers, 3 consultants and 3 trainees. Each observer read the radiographs on 2 separate occasions, independently, at least 2 weeks apart. Interobserver and intraobserver agreement and validity were analyzed, using weighted κ statistics. The mean κ value for interobserver agreement was found to be 0.849 (0.838-0.871) for consultants (almost perfect agreement) and 0.761 (0.707-0.836) for the trainees (substantial agreement). Intraobserver κ values ranged from 0.740 to 0.903, showing substantial to almost perfect agreement. Validity analysis of 23 type B cases revealed 79.710% agreement within B1, B2, and B3 subgroups with a mean κ value of 0.694 (0.670-0.741) (substantial agreement).

Preferential priming of alloreactive T cells with indirect reactivity.

The relative contributions of the direct and indirect pathways in alloimmune responses have not been fully elucidated. We report a novel murine TCR transgenic system that can simultaneously track the CD4-direct (CD4-d), CD4-indirect (CD4-i) and CD8-direct (CD8-d) pathways after transplantation. Using this system, we have observed a profoundly greater proliferation of CD4-i T cells relative to CD4-d and CD8-d T cells after transplantation. Furthermore, a much larger proportion of CD4-i T cells attain an effector phenotype. We also analyzed endogenous, wild-type T cells using enzyme-linked immunospot analysis. In naïve mice, T cells with indirect reactivity were undetectable, but T cells with direct reactivity were abundant. However, 10 days after skin or heterotopic heart transplantation, CD4-i T cells comprised approximately 10% of the CD4+ response. Consistent with increased priming of the CD4-i pathway, we observed that the CD4-i T cells were further enriched in the effector cells migrating to the allograft and in memory-like T cells persisting after rejection. Thus, priming of the CD4-i pathway is favored after transplantation, allowing a rare population to rapidly become a major component of the CD4+ T-cell response in acute allograft rejection. The generalizability of this observation to other models remains to be determined.

Spatiotemporal dynamics of CREB phosphorylation: transient versus sustained phosphorylation in the developing striatum.

The cAMP response element-binding protein (CREB) is a plasticity-associated transcription factor that can potentially integrate cAMP and calcium signals at the gene activation level. We tested for convergent Ser-133 phosphorylation of CREB via dopamine D1/D5 receptors and L-type calcium channels in organotypic cultures of neonatal striatum. We found such convergence only transiently. Sustained CREB phosphorylation by D1/D5 receptor and L-type channel agonists was targeted to opposite (striosome and matrix) cellular phenotypes. Subsequent expression of the CRE-containing gene, c-fos, matched the divergent patterns of sustained CREB phosphorylation, and both divergent patterns could be switched by inhibition of phosphatases, including calcineurin. Control of the duration of CREB phosphorylation may be a critical regulator of CRE-mediated gene expression by dopamine and calcium.

Sirtinol attenuates hepatic injury and pro-inflammatory cytokine production following trauma-hemorrhage in male Sprague-Dawley rats.

BACKGROUND: Although studies have demonstrated that sirtinol administration following adverse circulatory conditions is known to be protective, the mechanism by which sirtinol produces the salutary effects remains unknown. We hypothesized that sirtinol administration in male rats following trauma-hemorrhage decreases cytokine production and protects against hepatic injury. METHODS: Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). A single dose of sirtinol (1 mg/kg of body weight) or vehicle was administered intravenously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 levels in the liver and plasma alanine aminotransferase (ALT) concentrations were measured (n=6 Sprague-Dawley rats/group). RESULTS: Trauma-hemorrhage increased hepatic MPO activity, CINC-1, CINC-3, ICAM-1, and IL-6 levels and plasma ALT concentrations. These parameters were significantly improved in the sirtinol-treated rats subjected to trauma-hemorrhage. CONCLUSION: The salutary effects of sirtinol administration on attenuation of hepatic injury following trauma-hemorrhage are, at least in part, related to reduction of pro-inflammatory mediators.

Usefulness of human leucocyte antigen-B27 subtypes in predicting ankylosing spondylitis: Taiwan experience.

BACKGROUND: Genetic factors are clearly attributed to the susceptibility of ankylosing spondylitis (AS). The human leucocyte antigen (HLA)-B27 proved to be the very useful marker for diagnosing AS. The aim of this study was to determine the prevalence of HLA-B27 subtypes in Taiwan and to investigate whether these subtypes may be of help in predicting the diagnosis of AS. METHODS: A total of 314 patients with AS and a control group of 71 subjects positive for HLA-B27 detected by flow cytometry analysis were recruited for the study. HLA-B27 subtypes were confirmed by the polymerase chain reaction-sequence-specific primers and sequence-specific oligonucleotide probing. RESULTS: Four B27 alleles were identified: B*2704, B*2705, B*2706 and B*2707. HLA-B*2704 was the predominant allele. There were significant differences in the distribution of HLA-B27 subtypes between patients with AS and controls. Five of them who were homozygous for the B*2704 allele were solely found in AS group but not in controls. Statistical analysis showed that B*2704 was positively associated with AS, which suggested an increased possibility of having AS. Other HLA-B27 subtypes showed no strong correlation with AS. CONCLUSION: In the Taiwanese population, susceptibility to AS was determined by the presence of HLA-B*2704. Although B*2706 was reported to have a negative association with AS in Taiwanese, Thai and Chinese Singaporean populations, we report, in our study, two AS patients with B*2706 (0.6%). Disease heterogeneity suggests that other than genetic background, many pathogenic factors could be associated with AS. This may need to be investigated with a larger group of patients with AS and controls.

Blood Pressure Is Associated with Tea Consumption: A Cross-sectional Study in a Rural, Elderly Population of Jiangsu China.

OBJECTIVES: Tea has long been hypothesized to possess hypotensive effects. However, there is uncertainty regarding the association of tea consumption with arterial blood pressure (BP). We aimed to examine the association between tea consumption and BP components including systolic BP (SBP) and diastolic BP (DBP). DESIGN: Community-based, cross-sectional study of Suzhou City, Jiangsu Province, China. SETTING: Tea consumption has protective influence on BP and presence of hypertension. PARTICIPANTS: 4579 older adults aged 60 years or older from the Weitang Geriatric Diseases study. MEASUREMENTS: Detailed information regarding tea consumption was collected using a pre-designed questionnaire. BP components were measured at least 3 times with a minimum 5-minute interval, by well-trained research nurses. METHODS: Data of 4579 older adults (response rate: 82%) aged 60 years or older from the Weitang Geriatric Diseases study were included in the analysis and we estimated the relationship of tea consumption and BP using linear regression models and the association between tea consumption and risk of hypertension using logistic regression models. RESULTS: In linear regression models, higher tea consumption frequency was found to be associated with lower systolic BP values, after adjusting for the effect of age, sex, education level, lifestyle-related factors, and cardiometabolic confounding factors in overall (coefficient =-1.49, P=0.0003), normotensive (coefficient =-0.91, P=0.017) and participants without anti-hypertensive treatment (coefficient =-1.26, P=0.027). Significant inverse association between diastolic BP and frequency of tea consumption was also observed in the overall subjects (coefficient =-0.74, P=0.003). In multivariate logistic analyses, habitual tea drinking was inversely associated with presence of hypertension [odds ratio (OR)=0.79, P=0.011] , and there was a progressive reduction in risk associated with higher frequency of tea consumption (P for trend=0.011). CONCLUSION: Habitual tea consumption was found to be associated with lower values of components of BP and a reduced likelihood of having hypertension in older adults. Given the widespread consumption of tea throughout China and the world, together with the major cardiovascular disease risk, our findings have important implications for human health.

Ketorolac Tromethamine Spray Prevents Postendotracheal-Intubation-Induced Sore Throat after General Anesthesia.

Background. Postoperative sore throat is one of the major complaints of general anesthesia in the postanesthesia care unit. This prospective study investigated the preventive effect of ketorolac tromethamine spray in postendotracheal-intubation-induced sore throat after general anesthesia. Methods. Surgical patients undergoing general anesthesia with endotracheal intubation were recruited from a medical center. Patients were randomly assigned to group K (treated with 5% ketorolac tromethamine spray) or group D (treated with distilled water spray). Before intubation, each endotracheal tube was sprayed with the appropriate solution by physicians over the 20 cm length of the cuff. Each group comprised 95 patients fitting the inclusion and exclusion criteria for whom complete data sets were collected. The intensity of the sore throat was measured at 1, 3, 6, and 24 h after surgery, and data were compared. Results. The two groups had similar characteristics. Postoperative sore throat was significantly less frequent in group K than in group D (p < 0.001) and the pain intensity was significantly lower in group K than in group D at each time point (all p < 0.001). Conclusions. This study demonstrated that preanesthesia 5% ketorolac tromethamine spray could effectively decrease postendotracheal-intubation-induced sore throat in patients undergoing general anesthesia.

Accuracy and Trending of Continuous Noninvasive Hemoglobin Monitoring in Patients Undergoing Liver Transplantation.

BACKGROUND: Shift in large fluid volumes and massive blood loss during liver transplantation frequently leads to rapid changes in hemoglobin (Hb) concentration; thus, to ensure adequate tissue oxygenation, accurate and rapid determination of Hb concentration is essential in transplant recipients. The Radical-7 Pulse CO-Oximeter provides a noninvasive and continuous way to monitor Hb concentration (SpHb) in real time and is an ideal candidate for use during liver transplantation. In this study, we assessed the relationship between SpHb and total Hb (tHb) obtained from arterial blood samples during surgery. METHODS: Forty patients undergoing liver transplantation were enrolled in this study. tHb and time-matched SpHb were measured at 5 different phases throughout surgery. Paired SpHb and tHb levels were assessed using linear regression, Bland-Altman analysis, and the Critchley polar plot method. RESULTS: A total of 161 paired measurements with sufficient signal quality were analyzed. The correlation between SpHb and tHb was 0.59 (P < .001). Bland-Altman analysis revealed that a bias between SpHb and tHb was 2.28 g/dL, and limits of agreement (LoA) were from -0.78 to 5.34 g/dL. Trending analysis showed that 87% of data were located within the acceptable trending area, indicating that the trending ability was not satisfied. CONCLUSIONS: The Radical-7 Pulse CO-Oximeter was not sufficient to monitor Hb levels and trends during liver transplantation surgery in our cohort. In particular, in critical patients and in those with low Hb levels, invasive Hb measurement should be used for assessment.

Cardiac Output Assessed by the Fourth-Generation Arterial Waveform Analysis System Is Unreliable in Liver Transplant Recipients.

BACKGROUND: Liver transplant recipients often have violent hemodynamic fluctuation during surgery that may be related to perioperative and postoperative morbidity. Because there are some considerations for the risk of the pulmonary arterial catheter (PAC), the conventional invasive device for cardiac output (CO) measurement, a reliable and minimally invasive alternative is required. We validated the reliability of CO measurements with the use of a minimally invasive FloTrac system with the latest fourth-generation algorithm in liver transplant recipients. METHODS: Forty liver transplant recipients without atrial fibrillation, valvular pathology, or intracardiac shunt were recruited in this prospective, observational study. CO values measured by use of PAC with continuous thermodilution method (COTh) and FloTrac devices (COFT) were collected simultaneously throughout the operation for reliability validation. RESULTS: Four hundred pairs of CO data points were collected in total. The linear regression analysis showed a high correlation coefficient (73%, P < .001). However, the percent error between COTh and COFT was 42.2%, which is worse than the established interchangeability criterion of 30%. The concordance rates were calculated at 89% and 59% by 4-quadrant plot and polar plot analysis, respectively. Neither met the preset validation criteria (>92% for the 4-quadrant plot and >90% for polar plot analyses). CONCLUSIONS: Our study demonstrates that the CO measurements in liver transplant recipients by the latest FloTrac system and the PAC do not meet the recognized interchangeability criterion. Although the result showed improvement in linear regression analysis, it failed to display a qualified trending ability.

Regulation of multiple dopamine signal transduction molecules by retinoids in the developing striatum.

Increasing evidence based on pharmacological and genetic studies suggests that retinoid signaling plays an important role in developmental control of striatal neurons. In the present report, we screened for genes that might be regulated by retinoids in the developing striatum. We cultured tissue explants from the lateral ganglionic eminence (striatal primordium), and for regional comparison, its adjacent structures of the cerebral cortex and the medial ganglionic eminence in embryonic day 15 rat telencephalon. Using the ribonuclease protection assay, we found that both all-trans retinoic acid and 9-cis retinoic acid significantly up-regulated dopamine D1 receptor, heterotrimeric G protein olfactory, adenylyl cyclase type V and dopamine- and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein mRNAs in the lateral ganglionic eminence culture. By contrast, neither all-trans retinoic acid nor 9-cis retinoic acid significantly altered D1 receptor, heterotrimeric G protein olfactory, adenylyl cyclase type V and dopamine- and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein mRNAs in the cortical and the medial ganglionic eminence cultures except that D1 receptor mRNA was dramatically induced in the medial ganglionic eminence by retinoic acid treatments. To test whether the induction of multiple dopamine signaling molecules in the lateral ganglionic eminence was due to a general enhancement of neuronal differentiation by retinoic acid, we assayed the effects of retinoic acid on other differentiation markers, including glutamate decarboxylase 65, NR1 subunit of glutamate NMDA receptor and microtubule-associated protein-2. None of these genes were significantly altered by retinoic acid treatments in the lateral ganglionic eminence culture, indicating the specificity of gene regulation by retinoic acid signaling. As D1 receptor, heterotrimeric G protein olfactory, adenylyl cyclase type V and dopamine- and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein are important molecules involved in propagation of striatal dopamine neurotransmission, our study raises the hypothesis that retinoid signaling may coordinately activate the transcriptional program that is associated with the dopamine signaling pathway in developing striatal neurons. Such coordinate regulation by retinoids may be part of the mechanisms by which the complex yet highly organized neurochemical constituents of the striatum are established during development.

Expression of type VI adenylyl cyclase in the central nervous system: implication for a potential regulator of multiple signals in different neurotransmitter systems.

The aim of this study was to investigate the distribution of a calcium-inhibitable adenylyl cyclase type VI (type VI AC) in the central nervous system using an antiserum directed against the N-terminus of type VI AC. Our results indicate that type VI AC immunoreactivity is generally expressed in many brain regions with different levels of intensity. Most interestingly, the majority of the detected type VI AC immunoreactivity is present in cells of neuronal phenotype. Double immunostaining of type VI AC and markers of various neurotransmitter systems suggest that type VI AC might participate in regulation of the classical neurotransmitter systems and therefore appeared to play a very important role in the central nervous system.

Heterogeneous development of calbindin-D28K expression in the striatal matrix.

In the present study, we attempted to trace the development of the striatal matrix by analyzing the ontogenetic expression of calbindin-D28K (calbindin), a calcium binding protein selectivity expressed in medium-sized neurons of the matrix compartment of the mature rat's caudoputamen. The localization of calbindin was documented in a series of developing rat brains, as was the compartmental location of these cells relative to tyrosine hydroxylase (TH)-immunostained dopamine islands, sites of future striosomes. Medium-sized striatal neurons appeared in the striatum at embryonic day (E) 20, and from their first appearance, the calbindin-positive neurons had highly heterogeneous distributions. They first formed a latticework of patches and bands in a ventral region of the caudoputamen. By postnatal day (P) 7, this early calbindin-positive lattice had evolved into a mosaic in which circumscript pockets of low calbindin-like immunoreactivity appeared in more extensive calbindin-rich surrounds. With further development, the mosaic gradually encroached on all but the dorsolateral caudoputamen, a district that is calbindin-poor at adulthood. A special lateral branch of the striatal calbindin system was also identified, distinct from the rest of the calbindin-positive mosaic in several developmental characteristics. In the parts of the caudoputamen where the developing calbindin system and dopamine island system were both present, the dopamine islands invariably lay in calbindin-poor zones. Most dopamine islands, however, only filled parts of the corresponding calbindin-poor zones. Moreover, there were some calbindin-poor zones for which TH-positive dopamine islands could not be detected. Thus during development, calbindin was expressed in the extrastriosomal matrix of the striatum, but the matrix could be divided into calbindin-rich and calbindin-poor zones. In the calbindin-rich regions, there were patches of especially intense calbindin expression and zones of weaker expression. These results suggest that there is neurochemical heterogeneity in the striatal matrix during the prolonged developmental period in which the early calbindin-positive lattice expands to form the calbindin-positive matrix of the mature striatum. Surprisingly, calbindin expression in the matrix, although eventually distributed in strictly complementary fashion to striosomes, does not originate as a system complementary to dopamine islands. The prolonged disparity between the borders of dopamine islands and calbindin-poor zones, and the different spatiotemporal schedules of development of the islands and the calbindin gaps suggest instead that the final match between the borders of striosomes and surrounding matrix results from dynamic processes occurring early in postnatal development. Candidate mechanisms for the gradual adjustment of these borders are proposed.

Intrastriatal grafts derived from fetal striatal primordia: II. Reconstitution of cholinergic and dopaminergic systems.

Reconstitution of striatal cholinergic and dopaminergic systems was studied in intrastriatal grafts derived from embryonic day 15 rat striatal primordia and implanted into adult host rats in which unilateral ibotenic acid lesions had previously been made in the striatum. Histochemical, immunohistochemical, and ligand binding autoradiographic techniques were applied to analyze different constituents of these two systems and to study their locations relative to each other in grafts allowed to grow for 9-17 months following transplantation. For the cholinergic system, a modular organization was found in the striatal grafts with stains for choline acetyltransferase and acetylcholinesterase, respectively the synthetic and degradative enzymes for cholinergic neurons; by autoradiographic [3H]hemicholinium binding, specific for high affinity choline uptake sites associated with cholinergic terminals; and by autoradiographic [3H]pirenzepine binding, selective for M1 receptors. For the dopaminergic system, a comparable modular organization was found in the grafts by immunostaining for tyrosine hydroxylase, the catecholamine synthetic enzyme; by autoradiographic [3H]mazindol binding for dopamine uptake sites; and by [3H]SCH23390 binding for dopamine D1 receptors and [3H]sulpiride binding for dopamine D2 receptors. The results indicate that the distributions of the cholinergic and dopaminergic markers in striatal grafts are in close anatomical register. These markers for intracellular and membrane-associated components of the cholinergic and dopaminergic systems were preferentially localized in the acetylcholesterase-rich patches of the grafts in which cortical and thalamic fibers have also been found in striatal grafts, and in which output neurons projecting to the pallidum are located. This anatomical correlation suggests that the substrates for cholinergic-dopaminergic interactions typical of the normal striatum may be reinstated in the grafts both in relation to efferent neurons establishing connections with the host brain that are typical of normal striatofugal connections, and in relation to major afferent fiber systems from the host brain originating in regions known to project densely to the normal striatum. Accordingly, the cholinergic and dopaminergic systems in such grafts may regulate the functional influence of the grafts on the behavior of host animals.

Improved rapid photometric assay for quantitative measurement of PMN migration.

We developed an improved quantitative method to measure in vitro polymorphonuclear leukocyte (PMN) migration using an assembly consisting of a 96-well chamber, polycarbonate filter membrane, and a 96-well microtiter plate. The convenience in setup and counting of migrated cells using this method allows processing of 80 samples and 16 controls in a short assay time of only 2 h. The peroxidase contained in PMNs was used as a marker enzyme to determine the number of migrated cells. Peroxidase released from lysed migrated cells was detected with an enzymatic method utilizing o-dianisidine as substrate. Photometric measurement was performed with a conventional microtiter plate reader at a wavelength of 405 nm. Optical density readings obtained using the enzymatic assay correlated with the number of migrated cells in a linear fashion up to 1 x 10(5) cells/well. The sensitivity of the enzymatic assay was sufficient to determine cell counts as low as 500 PMNs. PMNs lost no measurable amounts of peroxidase during the migration assay when ZAS was used as the chemoattractant. A calibration method was developed to make corrections for variations in the peroxidase content of different cell preparations and changes in the peroxidase content of cells exposed to the chemoattractant. High speed, convenient handling, and the use of standard laboratory equipment result in low cost per assay and make this migration assay ideally suited to research and clinical applications.

Developmental restriction of the LIM homeodomain transcription factor Islet-1 expression to cholinergic neurons in the rat striatum.

LIM homeodomain transcription factors play crucial roles in determining diverse aspects of neuronal development both in vertebrates and invertebrates. In the present study, we studied the expression pattern of Islet-1 (Isl-1), a member of the LIM homeodomain protein family, in the rat striatum during development. The developmental expression of Isl-1 in the striatum is highly dynamic and complex in terms of spatial and temporal regulation. The reverse transcription-polymerase chain reaction and ribonuclease protection assays demonstrated that Isl-1 messenger RNA was expressed in the developing striatum. The immunocytochemical study of Isl-1 protein expression showed that there were prominent mediolateral and caudorostral Isl-1 gradients in the developing striatum. Numerous Isl-1-positive cells appeared in the medial mantle zone of the developing striatal proper, and they co-expressed the postmitotic neuronal marker, microtubule-associated protein 2. The numbers of Isl-1-positive cells were decreased from the medial to the lateral regions, so that there were only a few Isl-1-positive cells scattered in the lateral striatum. These scattered Isl-1-positive cells were doubly labeled with tyrosine kinase receptor A and choline acetyltransferase, which indicated that they were cholinergic neurons. The Isl-1 gradients were most prominent in the embryonic day 18 and 20 striatum. With increases of time, the Isl-1 gradients were gradually reduced, and the gradients disappeared by postnatal day 7. Despite the general down-regulation of striatal Isl-1, a few Isl-1-positive cells were sustained into the adult striatum in which Isl-1 was nearly exclusively expressed by all cholinergic neurons and vice versa. Our study suggests that Isl-1 is likely to be initially expressed by postmitotic cholinergic precursors and some, if not all, non-cholinergic precursors in the developing striatum. During the progression of striatal differentiation, Isl-1 is down-regulated in non-cholinergic cells, but is sustained in cholinergic cells. The developmental restriction of Isl-1 to cholinergic neurons in the striatum may represent a novel mechanism by which LIM homeodomain proteins specify specific cell types in the striatum during development.