Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources. cell separation methods/fluorescence in-situ hybridization/hereditary diseases/polymerase chain reaction/pregnancy     

Accumulation and defective β-oxidation of very long chain fatty acids in Zellweger''s syndrome, adrenoleukodystrophy and Refsum''s disease variants

The accumulation of very long chain fatty acids in plasma and skin fibroblasts was measured in at least four separate inherited disease states. Both the magnitude and the nature of the fatty acid changes reflected the clinical status of individual patients. In Zellweger's syndrome, and to a lesser extent in infantile Refsum's disease, there was an increase in 24:0, 26:0, 26:1, and a number of even longer chain fatty acids, while in the X-linked form of adrenoleukodystrophy these changes were less pronounced. Zellweger fibroblasts in culture took up lignoceric, phytanic and stearic acids and incorporated them into a variety of lipids in a manner comparable to control fibroblasts. However, these cells were unable to convert phytanic or lignoceric acid to CO2. Infantile Refsum's and X-linked adrenoleukodystrophy fibroblasts showed normal conversion of these acids to CO2. Normal fibroblast homogenates produced radioactive acetate from [1-14C] stearic and [1-14C] lignoceric acids indicating that both substrates were beta-oxidised under these conditions. Homogenates of fibroblasts from all patients patients with biochemical evidence of accumulation of very long chain fatty acids showed normal or near-normal stearic acid beta-oxidation, but were deficient in lignoceric acid beta-oxidation. Residual lignoceric acid beta-oxidation activity varied from approximately 15% in Zellweger syndrome up to 50% in X-linked adrenoleukodystrophy. It is postulated that the accumulation of very long chain fatty acids results from defects in peroxisomal beta-oxidation. In Zellweger's syndrome, and possibly in infantile Refsum's disease, it is probable that this defect is secondary to a primary abnormality affecting the structure and/or function of peroxisomes, while the primary defect in X-linked adrenoleukodystrophy may be confined to a pathway specific for the oxidation of very long chain fatty acids.     

Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity

During the process of bloodfeeding by Anopheles stephensi, mammalian latent transforming growth factor beta1 (TGF-beta1) is ingested and activated rapidly in the mosquito midgut. Activation may involve heme and nitric oxide (NO), agents released in the midgut during blood digestion and catalysis of L-arginine oxidation by A. stephensi NO synthase (AsNOS). Active TGF-beta1 persists in the mosquito midgut to extended times postingestion and is recognized by mosquito cells as a cytokine. In a manner analogous to the regulation of vertebrate inducible NO synthase and malaria parasite (Plasmodium) infection in mammals by TGF-beta1, TGF-beta1 regulates AsNOS expression and Plasmodium development in A. stephensi. Together, these observations indicate that, through conserved immunological cross talk, mammalian and mosquito immune systems interface with each other to influence the cycle of Plasmodium development.     

Eimeria necatrix: selection and characteristics of a precocious (and attenuated) line

A line of Eimeria necatrix with an abbreviated life cycle (i.e. a "precocious" line) was derived from the Houghton laboratory strain by repeated passage of the oocysts which were the first to be recovered from infections in chickens. The precocious line had a reproductive potential much lower than that of its parent strain and it was significantly less pathogenic. Its immunogenicity was, however, substantially retained. Selection for precocious development was accompanied by changes in the endogenous development of the parasite with an attendant reduction in its prepatent time.     

Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae.

Previous studies on Streptococcus pneumoniae have established that the pneumococcal proteins autolysin (N-acetylmuramyl-L-alanine amidase) and pneumolysin both contribute significantly to the virulence of the organism. In the present work, autolysin and a defined toxoid derivative of pneumolysin were tested, individually and in combination, for efficacy in a mouse model as antigens protecting against challenge with virulent, wild-type S. pneumoniae. While each antigen alone provided significant protection, the degree of protection was not increased when the antigens were administered together. In an additional experiment, mice were challenged with a genetically-modified mutant strain of pneumococcus unable to express active pneumolysin. Pre-immunization of such mice with autolysin failed to provide any significant protection against the challenge. The results of this study suggest that the most important contribution made by autolysin to the virulence of S. pneumoniae may be its role in mediating the release of pneumolysin from the pneumococcal cytoplasm during infection.     

Expression and characterization of a soluble rubella virus E1 envelope protein

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1ΔTm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1ΔTm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1ΔTm by the insect cells. The secreted E1ΔTm protein was purified from serum-free medium by onestep immunochromatography. The purified E1ΔTm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.     

Successful immune reconstitution in severe combined immunodeficiency despite Epstein-Barr virus and cytomegalovirus infections

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV), frequently found in the acquired immune deficiency syndrome (AIDS), have been suspected of contributing to the latter immunodeficiency. The ability of normal HLA-identical sibling bone marrow to reconstitute an 8-month-old infant with severe combined immunodeficiency infected with these two viral agents is of interest. After presentation with severe mucocutaneous candidiasis, cavitary pulmonary disease, nodular cutaneous lesions, and hepatic abscesses containing acid-fast organisms, immunologic studies revealed lymphopenia, 1-3% T cells, and no lymphocyte responses to mitogens. Prior to transplantation, the infant's blood B lymphocytes grew spontaneously in culture, suggesting they were infected with EBV. Indeed, an appropriate antibody response to EBV was detected at 2 months post-transplantation. At 3 weeks postgrafting, neutropenia and cholestatic jaundice developed without other signs of graft versus host disease. Liver biopsy demonstrated CMV but no EBV by DNA hybridization. There was evidence of T- and B-cell function by 2 weeks postgrafting, including vigorous in vivo and in vitro responses to candida. Although the blood lymphocyte T4:T8 ratio was inverted at 2 weeks, it reverted to normal by 6 weeks post-transplantation. All clinical disease resolved by 8 months and karotyping revealed all T and B lymphocytes to be XX. Thus, despite infections with both CMV and EBV, complete immunologic reconstitution was achieved in this, the most severe of all genetically determined immunodeficiency conditions, arguing against these viruses having a major role in the failure of bone marrow transplantation in AIDS.     

Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RT-PCR method

Of four solid carriers tested, Whatman paper No 1 was the best for storing blood and serum samples for the diagnosis of bovine viral diarrhoea (BVD) by means of viral RNA detection. The filter papers were impregnated with 10 microl of blood or serum, followed by air drying. Samples collected in this way from persistently infected animals had lost infectivity within a few days, but viral RNA could still be detected by RT-PCR for up to 6 months. When investigated by RT-PCR, 12 blood and 10 serum samples selected at random from animals persistently infected with BVD virus showed the same results whether samples had been spotted onto filters or examined directly from the liquid state. The filters spotted with blood or serum are convenient for storage and transport of samples to a diagnostic laboratory without the need for cooling. Sequencing of amplified RNA can be used subsequently for genetic typing.     

COVID-19 and Infant Hospitalizations for Seasonal Respiratory Virus Infections,New Zealand, 2020

In March 2020, a national elimination strategy for coronavirus disease was introduced in New Zealand. Since then, hospitalizations for lower respiratory tract infection among infants <2 years of age and cases of respiratory syncytial or influenza virus infection have dramatically decreased. These findings indicate additional benefits of coronavirus disease control strategies.