Women's Perspectives of Needs Surrounding Adverse Birth Outcomes: A Qualitative Assessment of the Neighborhood Impact of Adverse Birth Outcomes.

Objectives African Americans are two times more likely to suffer adverse birth outcomes (i.e., low birth weight, preterm birth, and infant mortality) when compared to all other ethnic groups and this pattern is no different for Douglas County, Nebraska, where the majority of African Americans in Nebraska reside. Our goal was to identify factors, as described by local women, that contribute to adverse birth outcomes in the predominantly African American community of Northeast Douglas County in Omaha, NE, to ensure that these women's voices were included in the development of interventions to improve their neighborhood's birth outcomes. The paper describes the results of a qualitative needs assessment of these women which will aid in the design and implementation of neighborhood-based solutions. Methods We brought together a group of women with varying levels of birthing experience, time spent living in the neighborhood, and overall community involvement. Individual in-depth, in person, and telephone interviews were used to collect participants' perceptions of birth outcomes, neighborhood resources for pregnant women, and neighborhood strengths and weaknesses. Results The needs assessment identified that, although women in this neighborhood have experience with adverse birth outcomes, these experiences are not discussed resulting in a lack of awareness of the wide spread racial disparities in birth outcomes and the efforts and resources to address this public health problem. Conclusions for Practice This study reveals the power of direct conversations with women impacted by adverse birth outcomes, as they must be primary partners in any efforts to improve birth outcomes.

Influence of in vitro maturation of engineered cartilage on the outcome of osteochondral repair in a goat model.

This study was designed to determine if the maturation stage of engineered cartilage implanted in a goat model of cartilage injury influences the repair outcome. Goat engineered cartilage was generated from autologous chondrocytes cultured in hyaluronic acid scaffolds using 2 d, 2 weeks or 6 weeks of pre-culture and implanted above hydroxyapatite/hyaluronic acid sponges into osteochondral defects. Control defects were left untreated or treated with cell-free scaffolds. The quality of repair tissues was assessed 8 weeks or 8 months post implantation by histological staining, modified O'Driscoll scoring and biochemical analyses. Increasing pre-culture time resulted in progressive maturation of the grafts in vitro. After 8 weeks in vivo, the quality of the repair was not improved by any treatment. After 8 months, O'Driscoll histology scores indicated poor cartilage architecture for untreated (29.7 ± 1.6) and cell-free treated groups (24.3 ± 5.8). The histology score was improved when cellular grafts were implanted, with best scores observed for grafts pre-cultured for 2 weeks (16.3 ± 5.8). As compared to shorter pre-culture times, grafts cultured for 6 weeks (histology score: 22.3 ± 6.4) displayed highest type II/I collagen ratios but also inferior architecture of the surface and within the defect, as well as lower integration with native cartilage. Thus, pre-culture of engineered cartilage for 2 weeks achieved a suitable compromise between tissue maturity and structural/integrative properties of the repair tissue. The data demonstrate that the stage of development of engineered cartilage is an important parameter to be considered in designing cartilage repair strategies.

Comparison of the absorption of calcium carbonate and calcium citrate after Roux-en-Y gastric bypass.

INTRODUCTION: Roux-en-Y gastric bypass (RYGB) restricts food intake. Consequently, patients consume less calcium. In addition, food no longer passes through the duodenum, the main site of calcium absorption. Therefore, calcium absorption is significantly impaired. The goal of this study is to compare two common calcium supplements in gastric bypass patients. METHOD: Nineteen patients were enrolled in a randomized, double-blinded, crossover study comparing the absorption of calcium from calcium carbonate and calcium citrate salts. Serum and urine calcium levels were assessed for peak values (C (max)) and cumulative calcium increment (area under the curve [AUC]). Serum PTH was assessed for minimum values (PTH(min)) and cumulative PTH decrement (AUC). Statistical analysis was performed using a repeated analysis of variance model. RESULTS: Eighteen subjects completed the study. Calcium citrate resulted in a significantly higher serum C (max) (9.4 + 0.4 mg/dl vs. 9.2 + 0.3 mg/dl, p = 0.02) and serum AUC (55 + 2 mg/dl vs. 54 + 2 mg/dl, p = 0.02). Calcium citrate resulted in a significantly lower PTH(min) (24 + 11 pg/ml vs. 30 + 13 pg/ml, p = 0.01) and a higher AUC (-32 + 51 pg/ml vs. -3 + 56 pg/ml, p = 0.04). There was a non-significant trend for higher urinary AUC in the calcium citrate group (76.13 + 36.39 mg/6 h vs. 66.04 + 40.82, p = 0.17). CONCLUSION: Calcium citrate has superior bioavailability than calcium carbonate in RYGB patients.

The effect of growth factor treatment on meniscal chondrocyte proliferation and differentiation on polyglycolic acid scaffolds.

The aim of this investigation was to determine the effect of growth factor treatment on ovine meniscal chondrocyte (OMC) proliferation in vitro and on the production of matrix proteins by OMCs grown within a polyglycolic acid (PGA) scaffold. Analysis of 72-h monolayer cultures using the mean transit time (MTT) assay revealed a greater increase in OMC numbers in the presence of platelet-derived growth factor (PDGF)-AB, PDGF-BB, insulin-like growth factor (IGF)-I, transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) than in untreated controls. In contrast, IGF-II and bone morphogenetic protein-2 had no effect on OMC proliferation at the concentrations tested. The growth factors that elicited the greatest proliferative response (PDGF-AB, PDGF-BB, TGF-beta1, and IGF-I) were subsequently tested for their ability to enhance OMC proliferation and differentiation within PGA scaffolds. Biochemical analysis revealed less glycosaminoglycan (GAG) production in the presence of all growth factors tested compared to untreated control samples. In contrast, all of the growth factors increased collagen type I production by OMCs within the scaffolds at day 20, and all except PDGF-BB resulted in an increase at day 39, when compared to appropriate control samples. With the exception of IGF-I, none of the growth factors tested had any significant effect on collagen type II production. Histological staining of sections from OMC-PGA scaffolds did not reveal any difference in GAG or collagen production between the treatment groups. However, immunohistochemical analysis demonstrated an increase in collagen type I expression and a decrease in collagen type II at day 39 in all growth factortreated constructs, concomitant with a high infiltration of cells. This suggests that PDGF-AB, PDGF-BB, TGF-beta1, and IGF-1 may be useful in future tissue engineering studies for promoting meniscal cell proliferation and differentiation within scaffolds.

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

Bi-zonal cartilaginous tissues engineered in a rotary cell culture system.

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.

Collagen in human aorta. Changes in the type III/I ratio and concentration of the reducible crosslink, dehydrohydroxylysinonorleucine in ascending aorta from healthy subjects of different age and patients with annulo-aortic ectasia.

The type III/I + III collagen ratio was studied in intima-medial samples of ascending aortas obtained from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and from control subjects, using electrophoretic analysis of cyanogen bromide peptides. The [3H]borohydride-reduced crosslinks of collagens were analysed by ion-exchange chromatography. Type III/I + III collagen ratios were twice as high in adult aortas as those found in skin samples of the same age. This ratio was lower in fetal and very young aortic samples and in 6-8 out of 12 pathological aortas (including one sample from a Marfan patient) when compared with adult controls. In contrast, the type III/I + III collagen ratio was high in fetal or very young skin and the values obtained from several patients did not differ from those of the control skin samples. In one pathological aorta out of six studied, the concentration of the reducible crosslink, dehydrohydroxylysinonorleucine, was higher than in controls, suggesting increased collagen synthesis or impaired maturation of collagen. These changes point to altered collagen metabolism in aortas of patients with annulo-aortic ectasia.

Characterization of the collagen of human hypertrophic and normal scars.

The collagen produced in response to an injury of human skin is initially stabilized by a cross-link derived from hydroxyallysine, and characteristic of embryonic skin. In normal healing there is a change over with time to the cross-link derived from allysine, which is typical of young skin collagen. In contrast, hypertrophic scars fail to follow the time-related changes of normal skin, but retain the characteristics of embryonic collagen, indicating a continued rapid turnover of the collagen. This is further supported by the high proportion of the embryonic Type III collagen present in hypertrophic scars.

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs.

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.

Mechanical properties of adult vertebral cancellous bone: correlation with collagen intermolecular cross-links.

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross-linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44-88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross-links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross-links, that is, hydroxylysyl-pyridinoline (HP), lysyl-pyridinoline (LP), and pyrrole cross-link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross-link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.

Dorsal-ventral differences in the glia limitans of the spinal cord: an ultrastructural study in developing normal and irradiated rats.

The dorsal and ventral surfaces of the lumbosacral spinal cord were examined in normal and irradiated postnatal rats. In normal rats between three and 13 days postnatal (DP), the glia limitans (GL) of the ventral surface was a more complex structure than the dorsal GL. This greater degree of complexity was manifested in a greater number of subpial astrocytes, a greater number of radial glial processes and a more advanced state in differentiation of its constituents. In rats irradiated at three DP and examined at 13 DP, the ventral GL remained intact and relatively unaffected by the radiation. In contrast, the dorsal GL was disrupted, and Schwann cells were seen within the dorsal funiculus. The ventral GL of the rat lumbosacral spinal cord is a more substantial structure than the dorsal GL during normal development. This factor alone may account for the integrity of the barrier properties of the ventral GL following radiation. However, our observations suggest that subpial astrocytes of the dorsal GL are more susceptible to radiation damage at three DP than the subpial astrocytes and radial glia of the ventral GL.

Schwann cell invasion of ventral spinal cord: the effect of irradiation on astrocyte barriers.

This study examines a radiation-induced invasion and spread of Schwann cells into ventral gray regions of the lumbar spinal cord. The prevalence of these cells within the gray matter and the time course of their appearance in the ventral spinal cord is quite different from the pattern of Schwann cell development in dorsal spinal cord reported previously. The focus is on 2 possible pathways, each involving astrocytic barriers, by which Schwann cells access the ventral gray matter. The first of these is the glia limitans covering the ventral surface of the spinal cord and the possibility that its integrity has been disrupted by the exposure to x-rays. Comparisons of the glia limitans, including its thickness, between irradiated and nonirradiated rats revealed that exposure to radiation did not result in any morphologically discernible alterations. The second barrier examined was the astrocytic covering of blood vessels. In irradiated animals the astrocyte processes that normally surround blood vessels were missing in some instances, and Schwann cells were observed at these sites. The difference between the dorsal and ventral occurrence of Schwann cells is that, whereas Schwann cells primarily follow axons, specifically dorsal root axons, to access the dorsal spinal cord, it appears that the presence of Schwann cells in the ventral portion of the spinal cord where their location is primarily in the gray matter is associated with the vasculature.

Biomechanical and biochemical study of a standardized wound healing model.

Standardized protocols were developed for use in a detailed investigation into the biomechanical and biochemical properties of a dermal wound healing model in the rat. The use of a rapid freezing method at -80 degrees C minimized the detrimental effects of freezing on the biomechanical properties of the tissue and also allowed for convenient inter-laboratory collaboration to be performed. The methodology described allowed for the simultaneous and reproducible measurement of tensile strength, collagen cross-linking and proteolytic enzyme activity. Increases in the tensile properties of the tissue with time were consistent with an active process of remodelling process as indicated by changes in the cross-link and enzyme profiles. Initially the granulation tissue was comparatively rich in the keto-imine cross-link hydroxylysino-keto-norleucine, which was later replaced by the aldimine cross-link dehydro-hydroxy-lysinonorleucine. The mature cross-link histidino-hydroxy-lysinonorleucine was not observed within the granulation tissue at any stage and was also absent in aged control skin. A peak of matrix metalloproteinase-9 activity was observed at early timepoints (48 hr) and then decreased rapidly to normal levels and is consistent with an acute inflammatory response. In contrast matrix metalloproteinase-2 activity peaked later (3 days) and then decreased gradually, consistent with its role as one of the predominant enzymes involved in the remodelling process. The results described validate the animal model used and emphasize its potential for use in combined biomechanical and biochemical studies of acute wound healing.

The development of monamine-containing neurons in the brain and spinal cord of the salamander, Ambystoma mexicanum.

The distribution of monoamine-containing neurons in the CNS of the developing and adult axolotl, Ambystoma mexicanum, has been investigated using the histochemical fluorescence technique of Falck and Hillarp combined with microspectrofluorimetry. The earliest catecholamine-containing neurons to be detected are located in the ventral ependymal zone of the spinal cord at the time of hatching (Stage 41). Between stages 43 and 46, catecholamine fluorescence can be detected in neurons in the following regions: nucleus preopticus, the hypothalamic-infundibular region, and the brain stem reticular formation. 5-HT-containing neurons are only observed in the midbrain raphe region and are first detected at stage 44. In contrast to these early monoamine fluorescing groups, catecholamine-containing neurons are not routinely detectable in the nucleus interpeduncularis until six months of age. All monoamine-containing neuronal groups detected in developing axolotls are also present in both sexes of the adult. However, the fluorescence intensity is less in monoamine-containing neurons observed in adults than in early developing subjects. All catecholamine-containing neuronal groups, with the exception of those located in the midbrain region (nucleus interpeduncularis, reticular zone) have fluorescent processes that contact the cerebrospinal fluid (CSF). The presence of CSF-contacting processes in the hypothalamic and spinal cord regions suggest that the CSF may act as a medium through which bioactive substances are transported from one brain region to another. Intense catecholamine fluorescence is observed in cells of the notochord prior to the detection of the monoamine-containing neurons in the CNS. A possible involvement of catecholamines in the inductive effects of the notochord during development is discussed.

The generation of neurons involved in an early reflex pathway of embryonic mouse spinal cord.

The generation of lateral motor neurons (LMNs), interneurons and dorsal root ganglion (DRG) neurons of the cervical mouse spinal cord has been investigated by [3H]thymidine autoradiographic techniques. This investigation has two main objectives: (a) to determine on which embryonic days these three neuronal populations are born, and (b) to investigate the possibility that the neurons comprising early reflex circuits might be formed by a retrograde temporal sequencing of generation. LMNs are the first neurons generated in the cervical spinal cord. They arise between E8.8 and E11.5, and approximately 90% of these cells are born within a 36-hour period between E9 and E10.5. The earliest time of origin for interneurons is on E9.5, and those cells which are generated between E9.5 and E10.5 cluster in two distinct regions of the adult spinal cord. One of these regions is the lateral portions of laminae IV through VI; this appears to be the location of many ipsilateral association neurons. DRG neurons begin to arise on E9.5 and their generation is completed by E14. There is a trend within the DRG population for large neurons to be born before small neurons. Those cells with diameters of 40 micron or greater reach their generation peak on E10.5, while those smaller than 40 micron arise in the greatest numbers on E12. The findings of other investigations have provided evidence for a retrograde sequence of synaptic closure in the formation of the early disynaptic forelimb reflex pathway. The temporal difference in synapse formation in the terminal fields of DRG and association neurons is discussed in terms of our observation that both of these populations appear to have similar generation times. We suggest that factors responsible for the delayed synaptic closure of DRG afferents include the greater distances and the degree of collateralization which these afferents must undergo in order to establish their terminal fields. Finally, we discuss the possibility that the temporal sequence of neuronal generation and factors involved with the growth of neurites combine to produce a retrograde sequence of synaptic closure in the early disynaptic forelimb reflex pathway of mouse spinal cord.

Cross-link profile of bone collagen correlates with structural organization of trabeculae.

Little is known regarding the mechanisms that govern the structural organization of cancellous bone. In this study, we compare the nature of the collagen in vertebral cancellous bone with the structural organization of its trabecular network. Cylindrical specimens of cancellous bone from vertebrae were obtained from nine autopsy subjects (ages 46-88). In each subject, eight pairs of corresponding samples were obtained from three levels in the spine and three areas within the vertebral body, leading to a total of 68 pairs of samples. The cylinders from one side were used for morphometry and the classical morphometrical parameters were obtained (BV/TV, bone volume fraction; Tb.Th, trabecular thickness; Tb.N, number; Tb.Sp, trabecular spacing) and strut analysis (TSL, total strut length; Nd, number of nodes; Fe, number of free-ends). The amount of osteoid bone was also quantified. The cylinders from the other side were powdered and used for collagen assessment, including the amount of collagen (% w/w), and its content in immature cross-links; such as hydroxylysinonorleucine (mol/mol of collagen) and dihydroxylysinornorleucine, as well as stable mature cross-links, such as hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), and the pyrrole cross-links. A random regression model was used to explore the correlations. None of the biochemical parameters correlated with the BV/TV except the ratio between immature and mature cross-links (eta(2) = 0.34, p < 0.05). There was no relationship between the amount of osteoid bone and the cross-link profile. However, the concentration of pyrrole and HP cross-links in the bone samples correlated with the structural organization of its trabeculae, but in an opposite direction. Hence, the pyrrole/HP ratio was a good predictor of Tb.Th, Tb.N, Tb.Sp, and TSL (eta(2) > 0.65 and p < 0.01) as well as Fe and star marrow space (eta(2) > 0.45 and p < 0.05). The cylinders from subjects with high pyrrole or low HP in their bone collagen had a relatively thick and simple structure. Those with low pyrrole and high HP had relatively thin trabeculae that were more numerous and spread over a complex network. The relative concentrations of the pyrrole and pyridinoline cross-links appear to reflect the structural organization of the trabeculae.

An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells.

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.

An improved multimembrane microassay for quantitating the motility of granulocytes and monocytes labeled with chromium-51.

Various modifications of the Boyden chamber chemotaxis assay have been used to screen patients for abnormalities in granulocyte or monocyte motility. In most cases, cell motility has been assessed by quantitating the fraction of cells that migrates from an upper chamber through a filter toward a lower chamber containing chemoattractant. Existing versions of the assay have several shortcomings. They are labor-intensive, require relatively large numbers of cells and lengthy incubation, or they require visual cell counting and do not permit assessment of cells which may drop off the filter into the attractant medium. We have improved the accuracy and efficiency of existing microchamber assays by using 51Cr-labeled cells to eliminate microscopic cell counting, shortening the incubation time, adjusting the assay sensitivity, and accounting for cells which drop off into the attractant well. The modified method uses Neuroprobe multiwell microchambers and two 10 microns polycarbonate filters with 3 microns pores on top of one 100 microns nitrocellulose filter. The optimal incubation period is 60 min, and the assay requires about one-fifth as many cells as the standard Boyden chamber methods. Cell drop-off can be measured accurately by harvesting the attractant wells with detergent, and the assay sensitivity is comparable to that of existing radiometric assays using large chambers. The data indicate that the range of chemotactic and random motility of normal granulocytes and monocytes measured in the modified assay system is comparable to that reported for studies which have used established motility assays.

In vivo class II transactivator expression in mice is induced by a non-interferon-gamma mechanism in response to local injury.

BACKGROUND: Tissue injury induces MHC class II expression, which could be important in the recognition of that tissue as an allograft. The class II transcriptional activator (CIITA) is the major regulator of basal and induced MHC class II expression and is essential for antigen presentation. The role of CIITA in the induction of class II by tissue injury is unknown. In this study, we examined CIITA induction in the course of acute ischemic or toxic renal injury in mice, including the role of interferon (IFN)-gamma and of the transcription factor, interferon regulatory factor (IRF)-1. METHODS: Kidneys were injured by ischemia or by gentamicin toxicity and were then studied for changes in gene expression using Northern blot, reverse transcriptase-polymerase chain reaction, radioimmunoassay, and tissue staining. We compared wild-type (WT) mice to IFN-gamma knockout (GKO) or IRF-1 knockout mice. RESULTS: Ischemic injury induced CIITA and class II expression in the kidney, in WT and GKO mice. Gentamicin injury also induced both CIITA and class II expression, independent of IFN-gamma, in WT and GKO mice. After ischemic injury, the induction of class II protein levels and CIITA and class II mRNA levels were induced, to a lesser degree, in IRF-1 knockout mice. CONCLUSIONS: These data indicate that CIITA is induced by tissue injury, and probably accounts for class II induction during tissue injury. CIITA induction by injury is largely IFN-gamma independent but requires IRF-1. The similarities of the pattern of CIITA and class II induction in ischemic and toxic injury suggest that this is a stereotyped response of injured tissue and not a consequence of a particular mechanism of injury.

Stretch receptor-associated expression of alpha 3 isoform of the Na+, K+-ATPase in rat peripheral nervous system.

Expression of the neuronal alpha(3) isoform of the Na(+),K(+)-ATPase (alpha(3) Na(+),K(+)-ATPase) was studied in the rat peripheral nervous system using histological and immunohistochemical techniques. Non-uniform expression of the alpha(3) Na(+),K(+)-ATPase was observed in L5 ventral and dorsal roots, dorsal root ganglion, sciatic nerve and its branches into skeletal muscle. The alpha(3) Na(+),K(+)-ATPase was not detected in nerve fibers in skin, saphenous and sural nerves. In dorsal root ganglion 12+/-2% of neurons were immunopositive for alpha(3) Na(+),K(+)-ATPase and all these neurons were large primary afferents that were not labeled by Griffonia simplicifolia isolectin B4 (marker of small primary sensory neurons). In dorsal and ventral roots 27+/-3% and 40+/-3%, respectively, of myelinated axons displayed immunoreactivity for alpha(3) Na(+),K(+)-ATPase. In contrast to the dorsal roots, strong immunoreactivity in ventral roots was observed only in myelinated axons of small caliber, presumably gamma-efferents. In the mixed sciatic nerve alpha(3) Na(+),K(+)-ATPase was detected in 26+/-5% of myelinated axons (both small and large caliber). In extensor hallicus proprius and lumbricales hind limb muscles alpha(3) Na(+),K(+)-ATPase was detected in some intramuscular axons and axonal terminals on intrafusal muscle fibers in the spindle equatorial and polar regions (regions of afferent and efferent innervation of the muscle stretch receptor, respectively). No alpha(3) Na(+),K(+)-ATPase was found in association with innervation of extrafusal muscle fibers or in tendon-muscle fusion regions. These data demonstrate non-uniform expression of the alpha(3) isoform of the Na(+),K(+)-ATPase in rat peripheral nervous system and suggest that alpha(3) Na(+),K(+)-ATPase is specifically expressed in afferent and efferent axons innervating skeletal muscle stretch receptors.