Noradrenergic projections to brainstem nuclei: evidence for differential projections from noradrenergic subgroups

Retrograde transport of the fluorescent tracer True Blue was used in combination with immunohistochemical staining of dopamine-beta-hydroxylase (a marker protein for noradrenergic neurons) to determine the origin of noradrenergic projections to three cranial nerve nuclei: 1) the motor nucleus of the trigeminal nerve, 2) the motor nucleus of the facial nerve, and 3) the spinal trigeminal nucleus pars interpolaris. Noradrenergic cells in the rat brainstem were divided into subgroups and their numbers were determined in serial sections stained with an antiserum to rat dopamine-beta-hydroxylase. Following tracer injections into the three brainstem nuclei, retrogradely labeled noradrenergic neurons were counted and the percentage of True Blue-labeled noradrenergic cells in each subgroup was calculated. Injections of tracer into the three cranial nerve nuclei resulted in distinctly different labeling patterns of noradrenergic cells. Of the total number of norepinephrine neurons projecting to the motor nucleus of the trigeminal nerve, 68% were observed within the A7 cell group; 75% of those innervating the motor nucleus of the facial nerve were found in the A5 cell group, and 65% of those projecting to the spinal trigeminal nucleus pars interpolaris were present in the locus ceruleus and subceruleus. These findings indicate that norepinephrine cells in the rat brainstem do not constitute a homogeneous population of cells but that several discrete systems can be identified that differ not only in topography but also in the terminal distribution of their axons. This combined retrograde transport-immunohistochemical study reveals a much higher degree of topographic order in the projections of norepinephrine neurons than has previously been recognized. The observation of differential projections of noradrenergic subgroups argues against the notion of a global influence of these cells over functionally diverse areas of the brainstem.     

The combination of absent thyroid peroxidase antibodies and high thyroid-stimulating immunoglobulin levels in Graves' disease identifies a group at markedly increased risk of ophthalmopathy.

Among Graves' Disease (GD) patients, we have observed an unexpectedly high prevalence of antithyroperoxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) negativity in those with severe ophthalmopathy. To study the possible role of thyroid autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO), TPOAb, TgAb, thyroid-stimulating immunoglobulin (TSI), and thyrotropin-binding inhibitory immunoglobulin (TBII) levels were measured, and the presence or absence of GO was assessed by a single observer in 100 consecutive patients with newly diagnosed, untreated GD who were nonsmokers. Ophthalmopathy was present in 43 patients. TSI levels (p = 0.001), and the prevalence of TPOAb-negativity (p = 0.002) were significantly higher in patients with ophthalmopathy compared to those without. Logistic regression analysis showed that TSI levels (p = 0.005) and the absence of TPOAb (p = 0.0025) were independent predictors of GO. No correlation between TBII or TgAb and eye disease was found. The prevalence of GO increased with each quartile of TSI levels. The prevalence was 20%, 36%, 52%, and 64% in the first, second, third and fourth quartiles of TSI, respectively. The odds ratio of GO (with 95% confidence intervals) when TSI levels were above the median level (1640%) was 3.6 (1.5-8.0), when TPOAb was negative it was 5.0 (1.7-14.4), and with both risk factors it was 36.6 (4.3-313.5). The prevalence of ophthalmopathy in this last group was 92.9%. The combination of negative TPOAb and high TSI levels appears to be associated with a markedly increased risk of clinically evident ophthalmopathy.     

Mechanisms of Interstitial Flow-Induced Remodeling of Fibroblast–Collagen Cultures

Interstitial fluid flow, critical for macromolecular transport, was recently shown to drive fibroblast differentiation and perpendicular cell and matrix alignment in 3D collagen cultures. Here we explore the mechanisms underlying this flow-induced cell and collagen alignment. Cell and matrix alignment was assessed from 3D confocal reflectance stacks using a Fast Fourier Transform method. We found that human dermal and lung fibroblasts align perpendicular to flow in the range of 5–13 μm/s (0.1–0.3 dyn/cm²) in collagen; however, neither cells nor matrix fibers align in fibrin cultures, which unlike collagen, is covalently cross-linked and generally degraded by cell fibrinolysis. We also found that even acellular collagen matrices align weakly upon exposure to flow. Matrix alignment begins within 12 h of flow onset and continues, along with cell alignment, over 48 h. Together, these data suggest that interstitial flow first induces collagen fiber alignment, providing contact guidance for the cells to orient along the aligned matrix; later, the aligned cells further remodel and align their surrounding matrix fibers. These findings help elucidate the effects of interstitial flow on cells in matrices and have relevance physiologically in tissue remodeling and in tissue engineering applications.     

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim: To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS). Methods: Ninety‐nine stools from 38 infants of median 27‐week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. Results: Standard microbiological culture identified a mean of two organisms (range 0–7), DGGE 12 (range 3–18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. Conclusions: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.