Triple paternal contribution to a normal/complete molar chimeric singleton placenta.

A comprehensive study of unusual cases of placental pathology may provide insight into mechanisms of normal human fertilization and early embryonic development by examining the exception to the rule. A gravida three para two 39-year-old woman was monitored by ultrasound from 16 weeks of gestation for cystic placenta. A female newborn was born at 36 weeks gestation. Pathologic examination of the partially cystic placenta revealed a singleton placenta comprised of 2/3 normal placenta and 1/3 complete hydatidiform mole, largely degenerated. Immunostaining for p57 was negative in stromal cells of the molar villi. Chromogenic in-situ hybridization revealed diploidy in both normal and molar parts. A total of 16 microsatellites were studied by short tandem repeat analysis, 11 of which were informative. The analysis revealed bipaternal molar tissue of dispermic origin. The paternal monospermic contribution to the normal part was different from that in the molar part, thus resulting in tripaternal contribution to the conceptus. A chimera is a single organism composed of two or more different populations of genetically distinct cells that originated from different zygotes (tetragametic) whereas mosaic is a mixture of two cell lines in one organism originating from one zygote. The possible mechanisms leading to the formation of chimeric/mosaic placenta in our case (one of the components being complete hydatidiform mole), including twinning, fusion at an early embryonic stage and diploidization of triploids, are discussed.

Naphthoquinone-tyrptophan reduces neurotoxic Aß*56 levels and improves cognition in Alzheimer's disease animal model.

An increasing body of evidence indicates a role for oligomers of the amyloid-ß peptide (Aß) in the neurotoxicity of this peptide and the pathology of Alzheimer's disease (AD). Several neurotoxic oligomeric forms of Aß have been noted ranging from the larger Amyloid ß-Derived Diffusible Ligands (ADDLs) to smaller trimers and dimers of Aß. More recently a dodecameric form of Aß with a 56 kDa molecular weight, denoted Aß*56, was shown to cause memory impairment in AD model mice. Here, we present for the first time a potential therapeutic strategy for AD that targets the early stages in the formation of neurotoxic Aß*56 oligomers using a modified quinone-Tryptophan small molecule N-(3-chloro-1,4-dihydro-1,4-dioxo-2-naphthalenyl)-L-Tryptophan (Cl-NQTrp). Using NMR spectroscopy we show that this compound binds the aromatic recognition core of Aß and prevents the formation of oligomers. We assessed the effect of Cl-NQTrp in vivo in transgenic flies expressing Aß(1-42) in their nervous system. When these flies were fed with Cl-NQTrp a marked alleviation of their Aß-engendered reduced life span and defective locomotion was observed. Finally, intraperitoneal injection of Cl-NQTrp into an aggressive AD mouse model reduced the level of the Aß*56 species in their brain and reversed their cognitive defects. Further experiments should assess whether this is a direct effect of the drug in the brain or an indirect peripheral effect. This is the first demonstration that targeted reduction of Aß*56 results in amelioration of AD symptoms. This second generation of tryptophan-modified naphthoquinones could therefore serve as potent disease modifying therapeutic for AD.

Living related liver transplant following bone marrow transplantation from same donor: long-term survival without immunosuppression.

We report long-term (seven yr) immunological tolerance in a 16-yr-old boy, to a liver allograft donated by his father following a bone marrow transplant at age 2.5 yr from the same donor. The bone marrow transplant was complicated by severe GVHD leading to liver failure and the ensuing need for a liver transplant, performed under planned avoidance of immunosuppression. At one wk post-transplant, although a liver biopsy was histologically compatible with acute rejection, favorable clinical and biochemical evolution precluded initiating immunosuppressive therapy, thus highlighting the need for caution when interpreting early histological changes so that administration of unnecessary immunosuppression can be avoided. Induction of tolerance in transplant recipients remains an elusive goal. In those patients who had received conventional bone marrow transplants and had endured the consequences of GVHD, development of macrochimerism may allow immunosuppression-free solid organ transplantation from the same donor.

Engraftment and development of human T and B cells in mice after bone marrow transplantation.

A model for human lymphocyte ontogeny has been developed in a normal mouse. Human bone marrow, depleted of mature T and B lymphocytes, and bone marrow from mice with severe combined immunodeficiency were transplanted into lethally irradiated BALB/c mice. Human B and T cells were first detected 2 to 4 months after transplantation and persisted for at least 6 months. Most human thymocytes (30 to 50 percent of total thymocytes) were CD3+CD4+CD8+. Human immunoglobulin was detected in some chimeras, and a human antibody response to dinitrophenol could be generated after primary and secondary immunization.

Patterned arrays of ordered peptide nanostructures.

Methods for the deposition of ordered nanostructures on various substrates are a key factor in nanotechnological devices. There is a special interest in the development of methods for the organization of organic nanostructures that are not compatible with some of the conventional fabrication methods. The unique chemical and physical properties of the peptide nanotubes make them excellent component in various devices and the useful application were already demonstrated in the case of biosensors. Here we demonstrate the ability to deposited aromatic dipeptide nanotubes using electron beam treatment of surfaces to control their wettability. The use of a low energy electron irradiation results in the formation of pre-defined surfaces with controlled level of wettability. This treatment allows the precise patterning of the organic tubular assemblies at high resolution. The differential wettability of the surface resulted in organization of the peptide assemblies according to the properties of the different areas of the surface. In the current work, we describe the use of wettability patterned surfaces for the control patterning of horizontal peptide nanotubes and nanospheres. Furthermore, lift-off lithography is used to make patterned arrays of peptide nano-forests, vertically aligned peptide nanotubes. In summary, the novel patterning techniques together with the unique properties of the peptide nanostructures represent an important step in the integration of these assemblies into functional nanosystems and devices.

A new approach to quantifying the EEG during walking: Initial evidence of gait related potentials and their changes with aging and dual tasking.

BACKGROUND: The electroencephalogram (EEG) can be a useful tool to investigate the neurophysiology of gait during walking. Our aims were to develop an approach that identify and quantify event related potentials (ERPs) during a gait cycle and to examine the effects of aging and dual tasking on these gait related potentials (GRPs). METHODS: 10 young and 10 older adults walked on a treadmill while wearing a wireless 20-channels EEG and accelerometers on the ankles. Each heel strike extracted from the accelerometers was used as an event to which the electrical brain activity pattern was locked. The subjects performed usual and dual task walking that included an auditory oddball task. GRPs amplitude and latency were computed, and a new measure referred to as Amplitude Pattern Consistency (APC) was developed to quantify the consistency of these GRP amplitudes within a gait cycle. The results were compared between and within groups using linear mixed model analysis. RESULTS: The electrical pattern during a gait cycle consisted of two main positive GRPs. Differences in these GRPs between young and older adults were observed in Pz and Cz. In Pz, older adults had higher GRPs amplitude (p = 0.006, p = 0.010), and in Cz lower APC (p = 0.025). Alterations were also observed between the walking tasks. Both groups showed shorter latency during oddball walking compared to usual walking in Cz (p = 0.040). In addition, the APC in Cz was correlated with gait speed (r = 0.599, p = 0.011) in all subjects and with stride time variability in the older adults (r = -0.703, p = 0.023). CONCLUSIONS: This study is the first to define specific gait related potentials within a gait cycle using novel methods for quantifying waveforms. Our findings show the potential of this approach to be applied broadly to study the EEG during gait in a variety of contexts. The observed changes in GRPs with aging and walking task and the relationship between GRPs and gait may suggest the neurophysiologic foundation for studying walking and for developing new approaches for improving gait.

Deterioration of specific aspects of gait during the instrumented 6-min walk test among people with multiple sclerosis.

Prolonged walking is typically impaired among people with multiple sclerosis (pwMS), however, it is unclear what the contributing factors are or how to evaluate this deterioration. We aimed to determine which gait features become worse during sustained walking and to examine the clinical correlates of gait fatigability in pwMS. Fifty-eight pwMS performed the 6-min walk test while wearing body-fixed sensors. Multiple gait domains (e.g., pace, rhythm, variability, asymmetry and complexity) were compared across each minute of the test and between mild- and moderate-disability patient groups. Associations between the decline in gait performance (i.e., gait fatigability) and patient-reported gait disability, fatigue and falls were also determined. Cadence, stride time variability, stride regularity, step regularity and gait complexity significantly deteriorated during the test. In contrast, somewhat surprisingly, gait speed and swing time asymmetry did not change. As expected, subjects with moderate disability (n = 24) walked more poorly in most gait domains compared to the mild-disability group (n = 34). Interestingly, a group × fatigue interaction effect was observed for cadence and gait complexity; these measures decreased over time in the moderate-disability group, but not in the mild group. Gait fatigability rate was significantly correlated with physical fatigue, gait disability, and fall history. These findings suggest that sustained walking affects specific aspects of gait, which can be used as markers for fatigability in MS. This effect on gait depends on the degree of disability, and may increase fall risk in pwMS. To more fully understand and monitor correlates that reflect everyday walking in pwMS, multiple domains of gait should be quantified.

Age-associated changes in obstacle negotiation strategies: Does size and timing matter?

INTRODUCTION: Tripping over an obstacle is one of the most common causes of falls among older adults. However, the effects of aging, obstacle height and anticipation time on negotiation strategies have not been systematically evaluated. METHODS: Twenty older adults (ages: 77.7±3.4years; 50% women) and twenty young adults (age: 29.3±3.8years; 50% women) walked through an obstacle course while negotiating anticipated and unanticipated obstacles at heights of 25mm and 75mm. Kinect cameras captured the: (1) distance of the subject's trailing foot before the obstacles, (2) distance of the leading foot after the obstacles, (3) clearance of the leading foot above the obstacles, and (4) clearance of the trailing foot above the obstacles. Linear-mix models assessed changes between groups and conditions. RESULTS: Older adults placed their leading foot closer to the obstacle after landing, compared to young adults (p<0.001). This pattern was enhanced in high obstacles (group*height interaction, p=0.033). Older adults had lower clearance over the obstacles, compared to young adults (p=0.007). This was more pronounced during unanticipated obstacles (group*ART interaction, p=0.003). The distance of the leading foot and clearance of the trailing foot after the obstacles were correlated with motor, cognitive, and functional abilities. CONCLUSIONS: These findings suggest that there are age-related changes in obstacle crossing strategies that are dependent on the specific characteristics of the obstacle. The results have important implications for clinical practice, suggesting that functional exercise should include obstacle negotiation training with variable practice of height and available response times. Further studies are needed to better understand the effects of motor and cognitive abilities.

Structure and orientation of the mammalian antibacterial peptide cecropin P1 within phospholipid membranes.

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly alpha-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospholipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cis positive transmembrane voltage difference. Taken together, the results further support a "carpet-like" mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.

Feto-feto-fetal transfusion syndrome in monozygotic monochorionic triamniotic triplets: vascular evaluation by a cast model.

A unique cast model of the placenta in a rare case of feto-feto-fetal triplet transfusion syndrome (FFFTTS) allowed the demonstration of why the transfusion syndrome developed in one fetus and not in the other two in that single placenta. The vasculature anatomy of a monochorionic triamniotic triplet placenta with FFFTTS of three healthy infants (one donor, two recipients) born in the 35th week of gestation was cast by means of dental casting materials. After the cast hardened, the tissue was corroded, revealing the cast blood vessels. The diameters and lengths of the chorionic blood and intraplacental vessels of the cast placenta were measured with a digital caliper. The cast revealed two artery-artery (A-A) anastomoses on the chorionic plate between the two recipients and the donor. Seven artery-vein (A-V) deep anastomoses connected only the arteries of the donor and the veins of the two recipients. The blood vessel connections among the fetuses allowed the evaluation of a pathologic case with its own control in a single placenta. From the vascular appearance, we speculate that the A-A anastomoses between the two fetuses protected them from developing blood transfusions, but that the A-V anastomoses contributed to their development.

HLA-A11 is associated with poor prognosis in childhood acute lymphoblastic leukemia (ALL).

A possible association between HLA antigens, susceptibility or resistance to leukemia, and responsiveness to treatment has been studied in 144 patients with childhood acute lymphoblastic leukemia (ALL) and compared to other prognostic factors, i.e. white blood cell (WBC) counts, age at onset, sex, ethnic origin, and cell surface markers. All sequentially newly diagnosed children (97) comprised the group for the prospective study (PSG) and were followed for 6 years. The group included 37 patients classified as T-ALL, 41 as CALLA+, 27 as NULL, 12 as B and pre-B, and 27 unclassified patients, who were diagnosed before 1980. During the follow-up period, 45 patients of the PSG died. Forty-seven patients designated long-term survivors (LTS) have been followed 6-20 years after diagnosis, having completed a 3-5 year course of anti-leukemia therapy, and having remained disease free thereafter. High WBC counts at diagnosis and T-cell-surface markers were associated with poor prognosis, as were enthnic origin and specific HLA antigens. Thus, there was one (1) a significant increase in HLA-A30 and a decrease in HLA B-14 in the PSG Jewish patients; and (2) a complete absence of HLA-ALL in LTS while, in the PSG, 8 of 9 HLA-All-positive patients died during the follow-up period. This suggests that HLA-All is associated with poor prognosis in childhood ALL.

Effect of cytosine arabinoside on differentiation of normal human bone marrow cells.

Human normal bone marrow cells were evaluated for alteration of differentiation after exposure for seven days to 10(-12)-10(-9) M cytosine arabinoside (ARA-C) in liquid culture. An increased number of induced cells had the morphologic appearance of mature monocytes-macrophages; they adhered to petri dishes, reacted positively to fluoride-sensitive naphthyl acetate esterase, and specifically bound My4 monoclonal antibody (MCAb). Assessment of phagocytosis and killing of Candida albicans (CA) by cultured monocytes-macrophages exposed to ARA-C demonstrated that treated cells had the same capacity to phagocytose and kill CA as did untreated cells. In semisolid culture, low doses of ARA-C did not affect myeloid colony growth. These studies indicate that ARA-C enhances monocytic differentiation of normal human bone marrow cells in liquid culture.

T6 positive cells in the peripheral blood of burn patients: are they Langerhans cells precursors?

Peripheral blood mononuclear cells of 14 patients suffering thermal injury were separated by affinity chromatography on peanut agglutinin (PNA) coupled to Sepharose macrobeads. The resulting PNA positive subset was 14% of the total mononuclear population. About 30% of these cells were found to coexpress T6(CD1), Ia-like and the myeloid differentiation antigens My4(CDw14) and Mo1(CD11). In comparison, the PNA+ subset from normal blood donors (about 5% of total mononuclear cells) contained mature monocytes that were found to be T6 negative. Electron microscopic studies using immunogold labeling showed that the T6 positive cells were slightly smaller than monocytes but larger than the classical lymphocytes and had common morphologic features with the Langerhans cells of the skin. Considering that patients suffering extensive damage of the epidermis require fast renewal of all skin elements, it is possible that the cells we identified in their peripheral blood are the precursors of the Langerhans cells of the skin en route from bone marrow to the epidermis.

A newly designed whole microplate automatic harvester for lymphocyte stimulation assays.

A unique automated sampling manifold designed to recover cells grown in standard 96 well microplates from their culture medium is described. Cells are recovered and washed on fiber glass filter discs. Incorporation of radioisotopes into cells, can then be measured by appropriate counting of the filter discs. Typical applications include termination of mixed lymphocyte cultures, assays of mitogen stimulation of lymphocytes and antigen-specific lymphocyte transformation and assays of interferon activity. The harvester can also be used in other biological systems where collection and washing of precipitates is desired.

Effect of systemic antibiotics on the microbial flora of the external ear canal in hospitalized children.

The effect of antibiotic therapy and hospitalization on the external ear canal flora was investigated in 131 children. Fifty-eight percent of the patients receiving antibiotic therapy had Gram-negative bacilli or yeasts in their external ear canal, compared with 17% of the patients who were hospitalized for ten days or longer and only 3% of the patients who were hospitalized for short periods. Antibiotic therapy is the major factor in determining the colonization rate of the external ear canal with potentially pathogenic flora. Children under 1 year of age seem to be the most susceptible group to this shift of flora.

Lymphocytes modulated by beta interferon express new non-HLA-A,B,C class I antigens.

Peripheral blood lymphocytes were cultured with recombinant beta interferon (INF-lymph). Three days in culture and 5 X 10(4) units/ml produced the best modulatory effect. Platelet absorbed alloantisera previously shown to contain non-HLA-A,B,C class I antibodies were tested with the INF-lymph. The serologic reactivity could be blocked by turkey sera to human B2-microglobulin but not by turkey anti-Ia-like. Absorption studies with INF-lymph obtained from donors expressing different HLA antigens indicated that the serologic reactivity is not due to HLA. Fourteen sera appeared to cluster in three groups, only one of which was found to be associated with HLA (HLA-A1). Antigenic modulation of INF-lymph obtained from HLA-A1+ individuals with HLA-A1 typing alloantisera eliminated subsequent lysis mediated by HLA-A1 alloantibodies and complement but not lysis with platelet absorbed antibodies included in the cluster associated with HLA-A1. These findings suggest independence from HLA-A1 and coexpression of HLA-A1 and A1-associated class I antigens. Family studies indicated that the reactivities segregated with HLA thus mapping it to chromosome VI. Similar to what we have described with PHA, beta interferon modulates lymphocytes and induces the expression of new class I differentiation antigens probably analogous to the murine Qa-T1a antigens.

Alteration of antigenic expression of human myeloid precursor cells (CFU-GM) in long-term cultures.

Comparison of myelopoiesis in continuous human bone marrow and cord blood cultures was performed. The presence of major histocompatibility complex (MHC) antigens on myeloid precursors (CFU-GM) from cord blood was confirmed using monoclonal antibodies (MoAb) defining class I (i.e., anti-beta 2-microglobulin (B2, MG1) anti-HLA-A,B heavy chain (W6/32), and anti-HLA locus B(4E), and class II (HLA-DR (7,2] antigens. The effect of long-term culture on the expression of these antigens was investigated. Our studies indicate that following prolonged in vitro culture there is a decrease in the expression of MHC antigens on CFU-GM. Our observations provide a rationale for considering the use of cord blood leukocytes subjected to long-term culture as an alternative to allogeneic hematopoietic cell transplantation.

Fractionation of human bone-marrow mononuclear cells with peanut agglutinin: phenotypic characterization with monoclonal antibodies.

Bone marrow mononuclear cells were fractionated by affinity chromatography on immobilized peanut agglutinin (PNA). The resulting PNA+ fraction represented 10% of the total cell number. Twenty percent of the cells within the PNA+ compartment coexpressed the T6, Ia, Mo1, and My4 differentiation antigens and possessed Fc and C3 receptors. The similarity in cell surface antigen phenotype led us to hypothesize that this subset may be a cellular precursor of dendritic cells found in the skin (Langerhans cells) or in the parenchimal organs of the body (D-cells).

A subset of human cord blood mononuclear cells is similar to Langerhans cells of the skin: a study with peanut agglutinin and monoclonal antibodies.

Mononuclear cells were fractionated from human cord blood by affinity chromatography on immobilized peanut agglutinin, as previously described (Rosenberg et al., Hum Immunol 7:67, 1983). The PNA+ subset was found to be composed mainly of a population of cells phenotyped as Ia+, T6+, M01+, and MY4+. The presence of mononuclear cells coexpressing these antigens was demonstrated by three techniques: double labeling immunofluorescence using FITC and rhodamine conjugated goat antimouse IgG; fluorescence activated cell sorter (FACS); and by direct counting (under the microscope) of cells stained by either individual or a combination of a variety of monoclonal antibodies. The PNA+ cells expressed cytoplasmic structures similar to Birbeck granules. In view of the fact that Langerhans cells of the skin share a similar phenotype and express Birbeck granules, we suggest that this subset may be the precursor of the Langerhans cells of the skin. In addition, these cells may also be the precursors of the dendritic cells found in the spleen, lymph nodes, thymus, and liver.

Characterization of human umbilical cord blood lymphocyte subsets fractionated on immobilized peanut agglutinin.

Human cord blood mononuclear cells from single donors were separated on minicolumns of peanut agglutinin (PNA) coupled to Sepharose beads to yield two fractions: unbound cells (PNA-, 78%) that were eluted with phosphate buffered saline, and bound cells (PNA+, 22%) eluted with 0.2 M D-galactose. The total yield was 86% and the cells were fully viable. There was no enrichment for macrophages or for surface immunoglobulin positive (B) cells in either the PNA+ or PNA- subset. Only 26% of the PNA+ lymphocytes formed rosettes with sheep red blood cells, in contrast to 53% of the PNA- lymphocytes. The response of the PNA+ cells to mitogens and allogeneic stimulation was considerably lower than that of the PNA- cells, while that of the latter was higher than the response of the unseparated cells. The average ratios of response of the PNA+ to PNA- cells were 0.25 for PHA, 0.20 for concanavalin A, 0.15 for pokeweed mitogen, and 0.15 In the mixed lymphocyte reaction. when tested with monoclonal antibodies to lymphocyte surface markers, it was found that the PNA+ fraction was depleted of mature T cells and enriched in Ia positive cells. Our data show that the low reactivity of human cord blood mononuclear cells may be ascribed to the presence of a subpopulation of lymphocytes which are immunologically immature. They also provide further evidence that in humans the PNA receptor is a marker for immature T or B lymphocytes.