Association between impedance to blood flow in umbilical arteries and infant survival in twin-to-twin transfusion syndrome.

OBJECTIVE: To evaluate infant survival according to the Doppler pattern of impedance to blood flow in the umbilical arteries (UAs) prior to laser surgery, in pregnancies with twin-to-twin transfusion syndrome (TTTS). METHODS: This was a retrospective study of women with a monochorionic diamniotic twin pregnancy who underwent laser surgery for TTTS between January 2012 and May 2018 at a single institution. Absolute intertwin difference in UA pulsatility index (DUAPI) was measured within 48 h prior to laser surgery. Twins with intermittent or persistent absent/reversed end-diastolic flow (EDF) in the UA (UA-EDF) were analyzed separately. Survival of both or at least one infant at birth and at 30 days postpartum was compared between pregnancies with an intertwin DUAPI of ≥ 0.4 and those with an intertwin DUAPI of < 0.4, as well as between fetuses with intermittent and those with persistent absent/reversed UA-EDF. Parametric and non-parametric tests were used for analysis. Regression analysis was performed to determine if intertwin DUAPI and intermittent or persistent absent/reversed UA-EDF were associated independently with infant survival, while controlling for gestational age at delivery, Quintero stage and other important confounding variables. RESULTS: Of 231 TTTS pregnancies that underwent laser surgery during the study period, UA Doppler information could be retrieved for 206 and delivery information was available for 184, which comprised the study population. Rates of double-twin survival at birth were significantly higher in pregnancies with an intertwin DUAPI of < 0.4 than in those with an intertwin DUAPI of ≥ 0.4 (83.9% (78/93) vs 50.0% (12/24); P < 0.001). Double-infant survival at birth was higher in pregnancies with intermittent compared to those with persistent absent/reversed UA-EDF (73.0% (27/37) vs 36.7% (11/30); P = 0.003). Regression analysis demonstrated that an intertwin DUAPI of < 0.4 was associated with increased survival of both twins at delivery (P < 0.001) and at 30 days postpartum (P = 0.002), as well as increased survival of at least one twin at delivery (P = 0.009). Similarly, intermittent absent/reversed UA-EDF was associated with increased survival of both twins at delivery (P = 0.007) and at 30 days after birth (P = 0.015). CONCLUSIONS: Evaluation of intertwin differences in UA impedance to blood flow as well as identification of intermittent or persistent absent or reversed UA-EDF prior to laser surgery could help in the prediction of double-infant survival at birth and to 30 days in twin pregnancies with TTTS. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Candidate genes for the hypoxic tumor phenotype.

In this study, we have analyzed changes induced by hypoxia at the transcriptional level of genes that could be responsible for a more aggressive phenotype. Using a series of DNA array membranes, we identified a group of hypoxia-induced genes that included plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor-binding protein 3 (IGFBP-3), endothelin-2, low-density lipoprotein receptor-related protein (LRP), BCL2-interacting killer (BIK), migration-inhibitory factor (MIF), matrix metalloproteinase-13 (MMP-13), fibroblast growth factor-3 (FGF-3), GADD45, and vascular endothelial growth factor (VEGF). The induction of each gene was confirmed by Northern blot analysis in two different squamous cell carcinoma-derived cell lines. We also analyzed the kinetics of PAI-1 induction by hypoxia in more detail because it is a secreted protein that may serve as a useful molecular marker of hypoxia. On exposure to hypoxia, there was a gradual increase in PAI-1 mRNA between 2 and 24 h of hypoxia followed by a rapid decay after 2 h of reoxygenation. PAI-1 levels were also measured in the serum of a small group of head and neck cancer patients and were found to correlate with the degree of tumor hypoxia found in these patients.

A tandem pair of Leishmania donovani cation transporting ATPase genes encode isoforms that are differentially expressed.

The second gene (ATPase 1b) of a tandem pair of cation transporting ATPases from Leishmania donovani was cloned and sequenced. The sequence of this gene was very similar to its upstream neighbor (ATPase 1a). Both genes contained a 2922 base open reading frame capable of encoding a protein of 974 amino acids. The genes differed at 34 nucleotide base positions, predicting 20 amino acid differences between the two peptides. These changes were clustered at the carboxy terminus with 15 changes occurring in the COOH-terminal 37 amino acids. However, these changes did not alter the highly charged nature of the carboxy terminus observed in ATPase 1a. The sequence was also conserved for 73 bases upstream of ATPase 1a and 1b but downstream conservation was limited to 15 bases beyond the termination codon. RNA from ATPase 1a was 5.2 kb and was present in both developmental forms of Leishmania. By contrast the ATPase 1b gene expressed a 5.75 kb transcript which was much more abundant in the amastigote form of Leishmania than in the promastigote form.

Prenatal diagnosis of the RhD fetal blood type on amniotic fluid by polymerase chain reaction.

OBJECTIVE: To assess the accuracy of a molecular assay for the determination of the fetal RhD status on amniotic fluid (AF)samples. METHODS: Amplification of DNA by polymerase chain reaction of a common sequence of the RhD and CE genes and of a unique sequence of the RhD gene was performed on AF directly or after DNA extraction. Samples of AF obtained from patients undergoing amniocentesis for standard obstetric indications were used for the study. RESULTS: Amplification of DNA was successful on 112 of 114 samples. One hundred four fetuses were found to be RhD positive and eight were found to be RhD negative. Serologic confirmation of the RhD blood type was available on 108 samples and DNA diagnosis was correct in all cases. CONCLUSION: Polymerase chain reaction can be used to determine accurately the fetal RhD blood type from AF samples.

A new method for isolating Trypanosoma brucei gambiense from sleeping sickness patients.

Low infectivity to laboratory mammals and low virulence make Trypanosoma brucei gambiense difficult to isolate and grow in amounts sufficient for biochemical characterization. We report the isolation of T.b. gambiense by feeding cryopreserved primary isolates to laboratory-reared Glossina morsitans morsitans, followed by rapid cultivation in vitro of procyclic forms dissected from infected tsetse fly midguts. This technique allows the characterization of hitherto unsampled populations and avoids selection due to long-term subpassage. Of 16 primary isolates from trypanosomiasis patients of the Fontem focus in Cameroon, 12 (75%) produced infections in tsetse whereas only 4 (25%) infected rats. Ten isolates were subsequently cultivated as procyclic forms in vitro; 2 failed to grow owing to bacterial contamination. In addition, 2 primary isolates from Côte d'Ivoire patients and a stock of low virulence from the Congo Republic were similarly grown. Only one primary isolate produced tsetse salivary gland infections, an observation consistent with the hypothesis that some populations of T.b. gambiense are intrinsically incompatible with G.m. morsitans.

Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon.

Antibodies to the variable antigen type (VAT) designated LiTat 1.3 are common in sera from parasitologically confirmed patients with gambian sleeping sickness. For this reason, LiTat 1.3 has been considered a suitable antigen for detecting Trypanosoma brucei gambiense in the Card Agglutination Test for Trypanosomiasis (CATT; Testryp-CATT, Smith Kline-RIT). However, surveys in the T.b. gambiense endemic focus of Fontem in Cameroon have suggested that expression of LiTat 1.3 might be rare or absent. We show here that the gene for LiTat 1.3 was indeed absent from some T.b. gambiense stocks isolated from this focus, and a LiTat 1.3-like gene was present in others. The divergent gene differed from the cloned version of LiTat 1.3. In addition, antibodies to LiTat 1.3 could not be detected in rabbits infected with either of the two kinds of T.b. gambiense from the Fontem area. We suggest that the absence of LiTat 1.3 expression in this focus may have important implications for the epidemiology and control of sleeping sickness, especially if heavy reliance is placed on the CATT.

Cytokine production by a megakaryocytic cell line.

The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1 alpha, 1 beta, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), interferon-alpha (INF-alpha), and basic fibroblast growth factor (bFGF). Additionally, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-alpha, beta, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-alpha was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-alpha, TNF-alpha, GM-CSF, SCF, IL-1 alpha, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-alpha, TNF-alpha, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.

Transforming a conservative company--one laugh at a time.

You wouldn't think of Brady Corporation as an obvious place in which to find a fun culture. This traditional Midwestern company, a manufacturer of industrial signs and other identification products, didn't even allow employees to have coffee at their desks until 1989. But when Katherine Hudson became CEO in 1994, she and her executive team determined that injecting some fun into the company's serious culture could create positive effects within the organization and contribute to increased performance and sales. In this article, Hudson distills her approach to overhauling Brady's culture into six principles of serious fun: More people than you might think are comfortable having fun at work; used with an awareness of cultural sensitivities, fun and laughter really are well-understood international languages; humor can help companies get through tough times; fun can be embodied in formal programs; spontaneous efforts at humor can also be effective; and encouraging fun should begin at the top. She richly illustrates each principle with examples. At Brady, getting people to loosen up and enjoy themselves has fostered a company esprit de corps and greater team camaraderie. It has started conversations that have sparked innovation, helped to memorably convey corporate messages to employees, and increased productivity by reducing stress, among other benefits. And the company has doubled its sales and almost tripled its net income and market capitalization over the past seven years. Brady's experience suggests that promoting fun within the workplace can lead not only to a robust corporate culture but also to improved business performance.

Immunodepression and the course of infection of a chronic Trypanosoma brucei infection in mice.

The relationships between course of infection, antigenic variation, and immunodepression of antibody responses to heterologous antigens have been investigated in mice chronically infected with Trypanosoma brucei. T. brucei Brunel University Trypanosomiasis (BUT) 64 produces a fluctuating parasitaemia lasting about 80 days and ending fatally. It is demonstrated that recurring peaks of parasitaemia are associated with the appearance of new variant antigenic types. At 21 and 31 days of infection, IgG responses to the heterologous antigen, sheep red blood cells (SRBC), are absent and IgM responses are less than 5% of normal. When a single dose of cyclophosphamide (300 mg/Kg) was injected into mice on day 31 of infection, the parasitaemia rose sharply in an uncontrolled fashion and the treated mice died in about 10 days. Cyclophosphamide, given in this way, is known to ablate antibody production completely but temporarily. It is therefore concluded that even though infected mice make extremely poor antibody responses to heterologous antigens, they are still capable of producing sufficient antibody to control peaks of parasitaemia associated with the emergence of new variant antigenic types. The significance of these findings is discussed in relation to recurrent hypotheses of trypanosome-associated immunodepression.

Extrachromosomal inheritance of susceptibility to trypanosome infection in tsetse flies. II. Susceptibility of selected lines of Glossina morsitans morsitans to different stocks and species of trypanosome.

Two lines of Glossina m. morsitans, selected for susceptibility and refractoriness to infection with a single stock of Trypanosoma congolense, have now been shown to be susceptible or refractory to different stocks of T. congolense and, also, to different stocks of T. b. brucei and T. b. gambiense. The mean midgut infection rates of the susceptible line obtained in different experiments with T. congolense, T. b. brucei and T. b. gambiense were, respectively, 66%, 56% and 55%; the corresponding mature (hypopharynx or salivary gland) infection rates were 37%, 23% and 0%. The highest mature infection rates obtained in individual experiments with susceptible flies were 65% (T. congolense) and 40% (T. b. brucei). Mean T. congolense and T. b. brucei midgut infection rates obtained with the refractory line were 29% and 33% respectively, the mature infection rates being 12% and 7%, all significantly lower than the corresponding rates in the susceptible line. Development of midgut infections in susceptible flies appears to take place irrespective of trypanosome stock or form. There is some evidence to suggest that higher infection rates can be obtained with flies infected and maintained on mammals rather than on in vitro feeding systems. Susceptible flies matured a significantly greater proportion of their midgut T. congolense and T. b. brucei infections than did the refractory line, which suggests that maturation of infections is influenced by the susceptibility status of the fly. However, the apparent inability of these flies to develop mature infections of a major T. b. gambiense genetic grouping suggests that maturation of infections established in the midgut is a phenomenon primarily associated with trypanosome genotype.

Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro.

A common V(D)J recombinase that recognizes a conserved recombination signal sequence (RSS) mediates the assembly of immunoglobulin (Ig) and T cell receptor (TCR) genes in B and T cell precursors. The rearrangement of particular Ig and TCR gene segments, however, is tightly regulated with respect to cell lineage and developmental stage. Using an in vitro system, we analyzed recombinase cleavage of RSSs flanking Ig and TCR gene segments in nuclei. We found that both the lineage-specificity and temporal ordering of gene rearrangement is reflected in the accessibility of RSSs within chromatin to in vitro cleavage.

Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins.

Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines. We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes. Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin. The K562, HEL, and CHRF-288-11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements. Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment. Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization. A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot. Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage.

Ultrasound-guided cardiac blood sampling of the rabbit fetus.

The object of the study was to determine whether cardiac sampling of the rabbit fetus could be successfully accomplished with minimal procedure-related loss. Pregnant rabbit dams were randomized to undergo ultrasound-guided fetal cardiac sampling in either the left or right uterine horn at 27 days of gestation; cesarean delivery was performed the following day. Liveborn pups from unsampled uterine horns underwent cardiac puncture immediately after birth. Fetal hematologic parameters were then compared to neonatal parameters. The acute fetal mortality from the sampled uterine horns was similar to that of the unsampled horns (3.6 vs. 4.5%). Fetal hematologic values were significantly higher than neonatal values with the exception of the reticulocyte count. This rabbit model offers a new approach for the evaluation of novel treatment modalities for hemolytic disease of the human fetus.

Ultrasound-guided blood sampling of rabbit fetuses.

A rabbit animal model for hemolytic disease of the newborn has been previously described. However, evaluating the effects of this disease was limited to histologic and hematologic examinations of liveborn kitlings. To assess the feasibility of in utero blood sampling, we performed ultrasound-guided cardiac sampling of 50 fetuses in 16 New Zealand White does on days 26 and 27 of gestation. The overall rate of successful sampling was 80%. The procedure-related mortality declined to 35% by the third phase of the study. The mean (+/- SD) hematocrit (%) and reticulocyte values (#/100 RBCs) on day 26 were 26.3 +/- 3.3 and 35.6 +/- 5.1, respectively; values on day 27 were 31.3 +/- 4.9 and 27.5 +/- 7.6. The results of this study suggest that hematologic data can be obtained from rabbit fetuses in the majority of cases with only moderate fetal loss.