A microscopical study of wound repair in the human placenta

In order to fulfill its many functions as the selective interface between maternal and fetal circulations it is imperative that the human placenta remains intact and in good operational order. That damage of some sort occurs during its short but extremely active life seems inevitable given the dynamic environment in which the placenta exists, and evidence has accumulated that disruption is indeed a regular event. The implications of such damage, one could speculate, may impact on functions such as transport and hormone secretion as well as mutual protection against attack by maternal and fetal immune systems. Consequently, it would seem a theoretical necessity for discontinuities in the placenta surface to be repaired as soon as possible. We have used a combination of ex vivo observation, in vitro modelling, immunohistochemistry and correlative microscopy to provide evidence for a wound response in the placenta and to begin dissecting the detail of how this may operate. Evidence for small lesions caused by fusion and subsequent tearing of the syncytiotrophoblast in vivo, as well as plugging of such wounds by underlying cells is shown. We also identify a putative role for migratory cytotrophoblasts in the healing of larger scale injuries and demonstrate that certain molecules, common to wound repair in other tissues, appear to be involved in placenta repair also. Taken together these results clearly show that the human placenta is capable of a degree of self-maintenance by activating what appears to be an endogenous wound healing mechanism.     

Widespread expression of MRP8 and MRP14 in human cerebral malaria by microglial cells

Recently, laser treatment of the prostate has been added to the urologist's armamentarium for the treatment of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). Until now, limited data on long-term outcome are available notwithstanding the fact that such information is crucial in determining the ultimate role of laser prostatectomy in the treatment of BPH. We now have 3-year data of a comparative study using the Urolase and Ultraline fiber in Nd:YAG sidefiring laser prostatectomy. The study was performed to compare laser prostatectomy using a pure coagulation (Urolase fiber) and a combination of a coagulation and vaporization (Ultraline fiber). In a period of 15 months, 93 men were randomized for laser treatment with the Ultraline fiber (N = 44) or the Urolase fiber (N = 49). Symptom scores, maximal uroflow, postvoiding residual volume, and sexual history were noted over a 3-year period. Adverse events and retreatments were also recorded. The mean postoperative catheterization time was 18 days, without significant difference between the two groups. After 3 years, we demonstrated a durable improvement in maximal flow rate, from 7.8 to 13.9 mL/sec in the Urolase group and from 7.9 to 13.6 mL/sec in the Ultraline group. In both groups, however, a considerable decrease in the maximal flow rate was noted after 3 years compared with 3 months after treatment, from 18.7 to 13.9 mL/sec in the Urolase group and from 20.0 to 13.6 mL/sec in the Ultraline group. The symptom scores showed marked and lasting improvement. The postvoiding residual urine volume became very low in the early postoperative period but did significantly increase after 3 years; nevertheless, it was still only 50% of the preoperative value. Although after 3 years, the maximal uroflow rate was still significantly improved compared with baseline, a considerable decrease was noted when compared with the early postoperative value. The same considerable and lasting improvement in subjective outcome (symptom scores) was seen in both groups. Although the Ultraline fiber also causes vaporization of prostatic tissue, no differences could be noted in the clinical outcome obtained with the two fibers.     

The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy

Synaptophysin, or p38, a polypeptide of molecular weight 38 kD, is a calcium-binding membrane protein found in synaptic vesicles of neurons and smooth surfaced vesicles of neuroendocrine cells. Six human neonatal and infant temporal bones were fixed in paraformaldehyde and glutaraldehyde, decalcified in EDTA and were than immunoreacted for synaptophysin (ICN Biomedicals) using the avidin-biotin reaction (ABC kit, Vector Labs). The tissue was then prepared for light microscopic surface preparation, radial sections of 5 microns, and serial section electron microscopy. At a light microscopic level, the inner spiral bundle, tunnel spiral bundle, upper tunnel crossing fibers and the base of outer hair cells were stained. At the base of outer hair cells, the immunoreactivity was seen to decrease from the base to the apex and from the first to third outer hair cells. At an electron microscopic level, immunoreactivity at the base of outer hair cells was limited to vesiculated efferent fibers. The degree of immunoreactivity between adjacent efferent fibers varied significantly. Immunoreactive vesiculated endings were also found in the supranuclear region of outer hair cells.     

Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage: an overview

Several microscopical techniques, such as high resolution light microscopy, Normaski microscopy, laser confocal and transmission electron microscopy, were used in a correlative morphological study of human osteoarthritic (OA) cartilage. Emphasis was made on the characterization of chondrocytes heterogeneity observed in this tissue. Novel findings were assessed in the morphological and immunocytological study of the chondrocytes organized in aggregates or "clones" typical of this degenerative disease, consisting of the modification of certain elements of the cytoskeleton that influence changes in the cell shape. Also, the presence of cilia and centrioles found in certain cell raised the question if chondrocytes are able to move and regroup as an alternative mechanism to mitosis in the formation of cell clusters or "clones." The presence of two types of secretory chondrocytes was observed and discussed. The use of a correlative approach of several microscopical techniques in a systematic morphological and immunocytological characterization of chondrocyte population within the fibrillated and nonfibrillated human osteoarthritic cartilage gave complementary information that could be important for a better understanding of the histopathogenesis of OA.     

Differences in submicroscopic structure of the extracellular matrix of canine femoral and tibial condylar articular cartilages as revealed by polarization microscopical analysis

The submicroscopic orientation patterns of sulfated glycosaminoglycan side chains of proteoglycan molecules and collagen fibrils were compared in different extracellular matrix areas of femoral and tibial articular cartilages of young adult beagle dogs using qualitative and quantitative polarization microscopic analytical methods. Paraffin sections were cut perpendicularly to the articular surfaces from the femoral and tibial condyles and stained. Picrosirius red F38 staining combined with an antecedent digestion with testicular hyaluronidase was used to enhance the optical anisotropy of collagen. Birefringence of sulfated glycosaminoglycan molecules was selectively amplified by a combination of carboxymethylation with CH3I and a subsequent staining with toluidine blue. The specimens were analysed in a polarization microscope equipped with compensator plates, and retardation values of birefringence were determined in territorial and interterritorial matrix areas of different zones using monochromatic plane polarized light. It was found that besides some similarities there were significant differences in the submicroscopic organization of extracellular matrix between femoral and tibial articular cartilages. Common structural features of the femoral and tibial cartilages were the sulfated glycosaminoglycans and collagen fibrils which were circularly oriented in the territorial matrix, and these components were longitudinally arranged within the trabeculae of the interterritorial matrix. Furthermore, the territorial matrix was a more densely packed structure than the interterritorial matrix. Our results revealed the following major differences between the two cartilages: The degree of orientation of sulfated glycosaminoglycans was higher in the femoral cartilage matrix areas as compared to the identical structures of the tibial cartilage; the collagen structure was more densely packed in the interterritorial matrix of the superficial and mineralization zones of the femoral cartilage than in the tibial cartilage, and except for the zone of mineralization, the degree of collagen orientation was higher in the territorial matrix of the femoral than the tibial cartilage. These findings suggest that the extracellular matrix of femoral condylar cartilage has a more densely packed molecular structure than the softer tibial cartilage matrix. This structural difference may have an influence on the pathogenesis of diseases involving articular cartilage.     

Antigenic variation of the Epstein-Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the fresh-water planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians.     

Mechanisms of directed attention in the human extrastriate cortex as revealed by functional MRI

A typical scene contains many different objects, but the capacity of the visual system to process multiple stimuli at a given time is limited. Thus, attentional mechanisms are required to select relevant objects from among the many objects competing for visual processing. Evidence from functional magnetic resonance imaging (MRI) in humans showed that when multiple stimuli are present simultaneously in the visual field, their cortical representations within the object recognition pathway interact in a competitive, suppressive fashion. Directing attention to one of the stimuli counteracts the suppressive influence of nearby stimuli. This mechanism may serve to filter out irrelevant information in cluttered visual scenes.     

Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CGS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.     

Dissociation of saccade-related and pursuit-related activation in human frontal eye fields as revealed by fMRI

The location of the human frontal eye fields (FEFs) underlying horizontal visually guided saccadic and pursuit eye movements was investigated with the use of functional magnetic resonance imaging in five healthy humans. Execution of both saccadic and pursuit eye movements induced bilateral FEF activation located medially at the junction of the precentral sulcus and the superior frontal sulcus and extending laterally to the precentral gyrus. These findings extend previous functional imaging studies by providing the first functional imaging evidence of a specific activation in the FEF during smooth pursuit eye movements in healthy humans. FEF activation during smooth pursuit performance was smaller than during saccades. This finding, which may reflect the presence of a smaller pursuit-related region area in human FEF than the saccade-related region, is consistent with their relative size observed in the monkey. The mean location of the pursuit-related FEF was more inferior and lateral than the location of the saccade-related FEF. These results provide the first evidence that there are different subregions in the human FEF that are involved in the execution of two different types of eye movements, namely saccadic and pursuit eye movements. Moreover, this study provides additional evidence that the human FEF is located in Brodmann's area 6, unlike the monkey FEF which is located in the posterior part of Brodmann's area 8.     

Cellular localization of insulin-like growth factor II mRNA in the human fetus and the placenta: detection with a digoxigenin-labeled cRNA probe and immunocytochemistry

IGF-II plays a major role in the regulation of human fetal growth and development. However, more extensive information on the cellular sites of IGF-II synthesis in the fetus would provide more insight into its role in fetal organogenesis. Thus we have determined the sites of IGF-II synthesis in 18-26-wk gestation human fetal tissues using in situ hybridization with a digoxigenin-labeled cRNA probe to localize IGF-II mRNA in fetal liver, kidney, adrenal gland, cerebral cortex, costal cartilage, skeletal muscle, and lung, and in placental tissue. In human fetal tissues it has to date been impossible to clearly assign IGF-II mRNA to epithelial cells of entodermal origin. Besides their already known localization in cell matrix and a variety of mesodermal cell types, strong IGF-II mRNA-positive signals were detected in epithelial cells in the liver (hepatocytes), bronchial and bronchiolar epithelium, undifferentiated renal tubular epithelium, mature glomerular epithelium, pelvic urothelium, and adrenal epithelial cells of the zona persistens. To identify the cellular location of immunoreactive IGF-II, we also performed immunocytochemical studies in tissues of the same fetuses. Every tissue studied except the cerebral cortex contained immunoreactive cells; however, immunostaining was generally weaker than in situ hybridization signals. Our data show that the distribution of IGF-II in human fetal tissue is much more widespread than hitherto thought. A digoxigenin-labeled detection system for IGF-II is more capable of detecting the cellular expression pattern of IGF-II than radioactive probes and is suitable for analysis of routinely prepared paraffin-embedded material.     

Three-dimensional appearance of Langerhans cells in human gingival epithelium as revealed by confocal laser scanning microscopy

Despite the reducing exposure to allogeneic blood in cardiac surgery, most of patients with anemia still require allogeneic blood. In this study, we have attempted to harvest the blood from cardiac patients with baseline hemoglobin levels below 11.0 g/dl using recombinant human erythropoietin (rHuEPO). 29 anemic patients undergoing cardiac surgery at our hospital between January 1994 and March 1997 were divided into two groups: 3 weeks' treatment with recombinant human erythropoietin (rHuEPO) and blood donation (group 1, n = 15) and iron supplementation alone (group 2, n = 14). There were no statistically significant differences among the two groups in patients characteristic and surgical data. No serious adverse events after phlebotomy were apparent in patients donating autologous blood. Patients in group 1 had significantly higher hemoglobin levels than patients in group 2 at 7 days before operation. The number of reticulocytes were increased at just before operation in group 1, whereas group 2 showed no significant increase. The estimated hemoglobin increase in group 1 were higher at 7 days and just before operation. In 75% of group 1, allogeneic blood transfusion could be avoided, while all patients in group 2 received allogeneic blood transfusion. This study suggests that the combination of rHuEPO administration and autologous blood donation would be beneficial for anemic patients in elective cardiac surgery. The use of rHuEPO should not be restricted to anemic patients.     

Structural organization of lymphatics in the monkey esophagus as revealed by enzyme-histochemistry

The fine structure and distribution of lymphatic vessels in the monkey esophageal wall were studied by light and electron microscopy using an enzyme-histochemical method. Identification of lymphatics was made by 5'-nucleotidase staining, whereas blood vessels were visualized by alkaline phosphatase staining. This technique revealed intramural lymphatic networks in the mucosa, submucosa, and myenteric layer of the esophagus. The organization of lymphatics in the esophagus basically conformed to that of the small intestine, although they showed certain distribution patterns peculiar to the esophagus. The lamina propria mucosae exhibited a double-layered lymphatic network, and lymphatic capillaries extended into its papillae. Despite their forming blind ends and being closed by endothelial cells, the lymphatics in the mucosal papillae were found to contain lymphocytes in their lumen. This suggests that free cells might penetrate the endothelium to enter these initial portions of the lymphatics.     

Cloning and characterization of Rep-8 (D8S2298E) in the human chromosome 8p11.2-p12

Assessed the effect of co-occurring versus not co-occurring internalizing and externalizing behavior problems on the reasons parents reported for clinical referral of their adolescent child. Reasons for referral were coded for 181 inpatient adolescents, and parent ratings of internalizing and externalizing behavior were obtained for a general population sample of 500 adolescents. Parents concurrently reported internalizing and externalizing behavior as reasons for referral less frequently (p < .0001) than would be expected given the correlation between these two domains in the general population sample. This suggests that the presence of externalizing problems may decrease parents' concern or awareness of internalizing problems, the presence of internalizing problems may decrease parents' concern or awareness of externalizing problems, or both. Implications for the clinical referral of adolescents and for informal parental efforts at helping their children with externalizing and internalizing problems are discussed.     

Regulation of progesterone biosynthesis in the human placenta by estradiol 17 beta and progesterone

The information available concerning the effects of chemotherapy administered during pregnancy is limited and consists of case reports and small series. A registry has been established at the National Cancer Institute, but there are currently only several hundred cases of neonates exposed to chemotherapy registered. All clinicians who care for women receiving chemotherapy during pregnancy should report those experiences to the National Cancer Institute to increase the data base. When chemotherapy is used during the embryogenesis period in the first trimester there is an increased rate of spontaneous abortion and major birth defects. The most toxic chemotherapeutic agents administered during pregnancy are methotrexate and aminopterin and should be avoided when possible, particularly during the first trimester. Pregnancy-related physiologic changes should be kept in mind when dosing and administering cytotoxic chemotherapy. The risk of fetal malformation when chemotherapy is administered during the second and third trimesters is probably not greater than background rate, but there may be a greater risk of stillbirth, fetal growth restriction, premature birth, and maternal and fetal myelosuppression. Breastfeeding should be avoided in women receiving chemotherapy.     

Persistence of the ten-nucleotide repeat in chromatin unfolded in urea, as revealed by digestion with deoxyribonuclease i

It is shown by enzymatic digestion of chromatin from rat liver or Guerin ascites tumour (GAT) that treatments, which abolish the 180 base pair repeat, as revealed by digestion with micrococcal nuclease (shearing in salt solutions of medium ionic strength, sonication, fixation with formaldehyde in the presence of 5 M urea), have little effect on the 10 nucleotide repeat, observed in deoxyribonuclease I digests.     

In vivo neurochemistry of the brain in schizophrenia as revealed by magnetic resonance spectroscopy

Magnetic resonance spectroscopy (MRS), an application of the methods of nuclear magnetic resonance (NMR), is a functional imaging modality that provides a view of localized biochemistry in vivo. A number of studies applying MRS to the neurochemistry of schizophrenia have been reported, which encompass a range of patient populations, states of medication, anatomic regions, nuclear species, and MRS techniques. A brief review of the history and methodology of NMR and MRS is presented. Comparison is made of MRS capabilities with other functional imaging modalities. Aspects of the neurochemistry of schizophrenia relevant to MRS studies are reviewed, as are the reported MRS studies involving patients with schizophrenia. Areas of consistent findings include decreased phosphomonoesters and increased phosphodiesters in frontal lobes, and decreases in the putative neuronal cell marker, N-acetylaspartate, in temporal lobes. Studies of neurotransmitters such as glutamate, gamma-aminobutyric acid, and glutamine have generated inconsistent results. New insights into alterations in neurochemistry in schizophrenia have been provided by MRS. Studies of neurotransmitters have future potential with improvements in field strength and in spectral editing techniques. MRS has the potential to measure brain medication levels and simultaneous effects on neurochemistry. MRS may assist in characterizing high-risk populations, and ultimately guide medication use.     

Serine protease inhibitors expressed in the process of budding of tunicates as revealed by EST analysis

To identify genes expressed during budding of the tunicate Polyandrocarpa misakiensis, we isolated and sequenced 624 clones from a directionally constructed cDNA library to prepare a catalog of expressed sequence tags (ESTs). A total of 233 ESTs matched genes of known sequence in the SwissProt database. About 24% out of them showed high similarity to ribosomal proteins, twice the value (12%) of pre-budding animals. ESTs involved in the respiratory chain also appeared with significant redundancy, suggesting that tunicate budding is accompanied by the enhancement of energy conversion as well as protein synthesis. Serine protease inhibitor (serpin) afforded another striking example of a gene that was highly expressed in the process of budding. The deduced amino acid sequences of five serpin cDNAs all had two consensus signatures of the Kazal's type of secretory protease inhibitor, one of which had an active site for trypsin and the other for elastase. In line with this, recombinant GST-fusion protein showed both trypsin and elastase inhibitor activities. In accordance with the EST analysis, the hemolymph taken from the budding stage showed the highest activity of trypsin inhibitor. We discuss a possible role that Polyandrocarpa serpins may play in bud development by counteracting trypsin-like serine protease, which could facilitate dedifferentiation of formative tissues.     

Distinct subpopulations in HaCaT cells as revealed by the characteristics of intracellular calcium release induced by phosphoinositide-coupled agonists

Both oximes and hydroxylamine (HYAM) are compounds with known oxidative capacity. We tested in vitro whether acetaldoxime (AAO), cyclohexanone oxime (CHO), methyl ethyl ketoxime (MEKO) or HYAM affect haemoglobin oxidation (into HbFe3+), formation of thiobarbituric acid reactive substances (TBARS), and glutathione (GT) depletion in human haemolysate, erythrocytes or blood. All these parameters are known to be related to oxidative stress. Glutathione S-transferase (GST) activity was measured as it may be affected by oxygen radicals. All three oximes caused a low degree of HbFe3+ accumulation in erythrocytes. This was higher in haemolysates indicating that membrane transport may be limiting or that protective mechanisms within erythrocytes are more effective. HbFe3+ accumulation was lower for the oximes than for HYAM. AAO and HYAM caused TBARS formation in blood. For HYAM this was expected as free radicals are known to be generated during HbFe3+ formation. Free radical generation by AAO and HYAM in erythrocytes was confirmed by the inhibition of GST. For the other two oximes (CHO and MEKO) some special effects were found. CHO did inhibit erythrocyte GST while it did not cause TBARS formation. MEKO was the least potent oxime as it caused no TBARS formation, little HbFe3+ accumulation and little GST inhibition in erythrocytes. However, GT depletion was more pronounced for MEKO than for the other oximes, indicating that glutathione conjugation occurs. TBARS formation, GT depletion and GST modulation caused by the oximes and HYAM were also tested in rat hepatocytes. However, no effects were found in hepatocytes. This suggests that a factor present in erythrocytes is necessary for free radical formation. Studies with proposed metabolites of the oximes (i.e. cyclohexanone, acetaldehyde or methylethyl ketone) and addition of rat liver preparations to the erythrocyte incubations with oximes, suggest that metabolism is not a limiting factor in erythrocyte toxicity.