Identification of aceNKPs, a committed common progenitor population of the ILC1 and NK cell continuum.

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

Group-2 innate lymphoid cell-dependent regulation of tissue neutrophil migration by alternatively activated macrophage-secreted Ear11.

Type-2 immunity is characterised by interleukin (IL)-4, IL-5 and IL-13, eosinophilia, mucus production, IgE, and alternatively activated macrophages (AAM). However, despite the lack of neutrophil chemoattractants such as CXCL1, neutrophils, a feature of type-1 immunity, are observed in type-2 responses. Consequently, alternative mechanisms must exist to ensure that neutrophils can contribute to type-2 immune reactions without escalation of deleterious inflammation. We now demonstrate that type-2 immune-associated neutrophil infiltration is regulated by the mouse RNase A homologue, eosinophil-associated ribonuclease 11 (Ear11), which is secreted by AAM downstream of IL-25-stimulated ILC2. Transgenic overexpression of Ear11 resulted in tissue neutrophilia, whereas Ear11-deficient mice have fewer resting tissue neutrophils, whilst other type-2 immune responses are not impaired. Notably, administration of recombinant mouse Ear11 increases neutrophil motility and recruitment. Thus, Ear11 helps maintain tissue neutrophils at homoeostasis and during type-2 reactions when chemokine-producing classically activated macrophages are infrequently elicited.

An engineered Plasmodium falciparum C-terminal 19-kilodalton merozoite surface protein 1 vaccine candidate induces high levels of interferon-gamma production associated with cellular immune responses to specific peptide sequences in Gambian adults naturally exposed to malaria.

The 19-kDa C-terminal region of merozoite surface protein 1 (MSP1(19)), a major blood stage malaria vaccine candidate, is the target of cellular and humoral immune responses in humans naturally infected with Plasmodium falciparum. We have previously described engineered variants of this protein, designed to be better vaccine candidates, but the human immune response to these proteins has not been characterized fully. Here we have investigated the antigenicity of one such variant compared to wild-type MSP1(19)-derived protein and peptides. Gambian adults produced both high T helper type 1 (Th1) [interferon (IFN)-γ] and Th0/Th2 [interleukin (IL)-13 and sCD30] responses to the wild-type MSP1(19) and the modified protein as wells as to peptides derived from both forms. Response to the modified MSP1(19) (with three amino acid substitutions: Glu27Tyr, Leu31Arg and Glu43Leu) relative to the wild-type, included higher IFN-γ production. Interestingly, some peptides evoked different patterns of cytokine responses. Modified peptides induced higher IL-13 production than the wild-type, while the conserved peptides P16 and P19 induced the highest IFN-γ and IL-13 and/or sCD30 release, respectively. We identified P16 as the immunodominant peptide that was recognized by cells from 63% of the study population, and not restricted to any particular human leucocyte antigen D-related (HLA-DR) type. These findings provide new and very useful information for future vaccine development and formulation as well as potential Th1/Th2 immunmodulation using either wild-type or modified protein in combination with their peptides.

Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum.

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.

Follow up of workers previously exposed to silver solder containing cadmium.

OBJECTIVES: To study longitudinal biological monitoring data on urinary and blood cadmium collected in a small cohort of nine workers who had been brazing for several years with solders containing cadmium. METHODS: Cadmium was measured by neutron activation analysis in livers and kidneys, and estimates of renal function were carried out in 1983 and 1995. During the intervening period exposure to cadmium was dramatically reduced by local exhaust ventilation control and substitution of the solder containing cadmium. RESULTS: From urinary protein measurements there was evidence within the group of increasing renal tubular damage over the 12 year period, even though exposure to cadmium was dramatically reduced over this period and almost eliminated by 1995. There was no evidence from serum creatinine of decreasing glomerular filtration rate, and the renal tubular handling of calcium, phosphate, or urate had not worsened significantly. Blood and urinary cadmium concentrations reduced significantly over the 12 year period but were still substantial in 1995. Blood cadmium concentrations tended to reflect cadmium body burden in 1995 when exposure had been low for several years, and decreased most significantly during 1983-90. By contrast urinary cadmium concentrations only decreased significantly from about 1990 onwards. Urinary cadmium was not significantly correlated with liver or kidney cadmium concentration in either 1983 or 1995. This may be due to the level of tubular dysfunction in the cohort. Calculated cumulative excretion of cadmium over the 12 year period was substantially greater than the loss of cadmium measured in livers and kidneys and the derived loss in body burden. Reasons for this are discussed. It is possible that in cohorts, where renal damage is apparent, urinary concentrations reflect a substantial component of current exposure rather than stored body losses. CONCLUSIONS: The data reinforce the concept that blood cadmium concentrations may not always reflect recent exposure, but may reflect body burden derived from historical exposure depending on the degree of current exposure; and that the decline in urinary and blood cadmium measurements after removal from, or reduction in, exposure will be slow and depend on the historical body burden.

Longitudinal measurements of the cadmium burden of 'jig solderers' using IVNAA.

The liver and kidney cadmium burdens of a population of 10 'jig solderers' were measured in 1983 and 1995 using similar IVNAA measuring systems. Cadmium exposure ceased in 1985. No significant variation in kidney content was observed. The mean body burden had fallen by 18%, due to a decrease of mean liver content of 35%.

The effect of oestradiol implants on regional and total bone mass: a three-year longitudinal study.

OBJECTIVE: Although there is evidence from cross-sectional studies that percutaneous oestrogen administration protects against menopausal bone loss, few longitudinal data are available. We have examined the effect of 3 years' treatment with percutaneous oestradiol on total body calcium, spinal trabecular bone mineral density and radial bone mineral content in post-menopausal women. DESIGN AND PATIENTS: Twenty-nine post-menopausal women, aged 37-55 years, who had undergone hysterectomy and had experienced the onset of menopausal symptoms within the previous 2 years, were studied before and for 3 years during hormone replacement with oestradiol implants, given at approximately 6-monthly intervals. MEASUREMENTS: Total body calcium was measured by prompt gamma neutron activation analysis, spinal trabecular bone mineral density by quantitative computed tomography and radial bone mineral content by single-photon absorptiometry. RESULTS: There was a significant increase in the mean total body calcium, spinal trabecular bone mineral density and radial bone mineral content over the 3 years of the study. The mean (+/- SEM) percentage change per annum was +2.4% (+/- 0.8) for total body calcium (P < 0.01), +3.3% (+/- 0.6) for spinal trabecular bone mineral density (P < 0.001) and +1.2% (+/- 0.6) for radial bone mineral content (P < 0.05). CONCLUSIONS: Percutaneous oestradiol replacement therapy prevents menopausal bone loss and is associated with a sustained and significant increase in total body calcium, spinal trabecular bone mineral density and radial bone mineral content over a 3-year treatment period. Oestradiol implants thus have skeletal effects comparable to those of oral or transdermal oestrogens.

The development of a technique to measure bone aluminium content using neutron activation analysis.

The accumulation and toxicity of aluminium in patients with chronic renal failure is a well recognized hazard, and there is a need for a non-invasive technique to assess Al tissue load in these patients. The technique of in vivo neutron activation analysis, using a thermal neutron beam from a reactor, has been employed by previous workers, who measured Al in the hand with a detection limit of 0.4 mg for a dose equivalent of 20 mSv. However, the application of this technique is restricted by the very limited availability of nuclear reactors. We report the modification of an existing 252Cf-based instrument and construction of a shielded, high-efficiency counting system for the in vivo measurement of Al in the hand. Phantoms containing tissue-equivalent solutions of Ca, P, Na and Cl with various Al loadings were used for validation of the technique. The Al/Ca ratio in the hands of seven patients with renal failure was measured using a cyclic activation technique to compensate for the relatively low neutron output of the 252Cf source, and a detection limit of approximately 2.2 mg Al was achieved for a dose equivalent of 36 mSv. The results were compared with the Al content of iliac crest bone biopsy specimens measured using electrothermal atomic absorption spectrophotometry.

Total body calcium in patients with inflammatory bowel disease: a longitudinal study.

1. Serial measurements of total body calcium have been made by prompt gamma-neutron activation analysis in 13 patients with inflammatory bowel disease over a mean period of 23 months. Changes in spinal trabecular bone mineral density and radial shaft bone mineral content were also assessed by using quantitative computed tomography and single photon absorptiometry, respectively. 2. The mean annual decreases (95% confidence intervals) were: total body calcium, 7.8% (-12.0 to -3.7%; P less than 0.001); spinal trabecular bone mineral density, 2.5% (-5.0 to +0.1%; 0.05 less than P less than 0.1), radial bone mineral content, 2.1% (-3.4 to -0.8%; P less than 0.01). 3. No significant correlations were found between rates of change of the three variables. However, there were significant positive correlations between the baseline values for total body calcium and radial bone mineral content (r = 0.638, P less than 0.05), spinal bone mineral density and radial bone mineral content (r = 0.854, P less than 0.01), and total body calcium and spinal bone mineral density (r = 0.876, P less than 0.001). 4. These results demonstrate rapid decreases in total body calcium in patients with inflammatory bowel disease which, in conjunction with the significant decrease in radial shaft bone mineral content, indicate increased rates of cortical bone loss. Whilst values for bone mass at different skeletal sites showed positive correlations within individuals, no relationship was found between the rates of change in bone mass at these sites. 5. The rapid bone loss observed in some subjects emphasizes the importance of early detection of osteoporosis by bone densitometry and the need for effective prophylactic measures to be established in this group of patients.

In vivo measurements of cadmium and lead in occupationally-exposed workers and an urban population.

This paper reports the preliminary findings of a survey of lead and cadmium body burdens in a nonoccupationally exposed population in Swansea, Wales, using the techniques of in vivo neutron activation and X-ray fluorescence analysis. Some measurements on an occupationally cadmium-exposed group are also included. The results confirm the association between cadmium and smoking and bone lead and age. The in vivo measurements demonstrate a degree of comparability with other data, which supports the further detailed analysis of the relationships between body burden and exposure, on the one hand, and possible health effects on the other.

Determination of total body calcium by prompt gamma neutron activation analysis: absolute in vivo measurements.

Sequential TBCa measurements using the prompt gamma ray technique are firmly established and a number of patient groups are under investigation. A technique has been proposed for the absolute measurement of TBCa based upon the use of TBCl as an internal standard. Further work is needed to establish the validity of the method, but the initial results are encouraging.

A feasibility study for the in vivo measurement of beryllium by photonuclear activation.

Diagnosis of the beryllium-induced disease, berylliosis, is often difficult and always requires that the presence of the metal in tissue be demonstrated. The feasibility of developing an in vivo method of measurement, which exploits the uniquely low photonuclear reaction threshold of 1.67 MeV in beryllium, has been investigated. Suitable photon sources and detector systems were assessed, both experimentally by phantom studies and theoretically by use of a Monte-Carlo neutron transport code. It is concluded that by using a filtered source of 124Sb for bilateral irradiation of the chest, and an array of twenty BF3 counters, beryllium could be measured to an accuracy of 0.33 mg per lung, which corresponds to a 2 SD detection limit of 0.67 mg, for a skin dose of 50 mGy delivered within a period of 90s. Such a facility would be capable of contributing to the aetiology of the disease in a large proportion of cases, but the wider use of the method for screening exposed workers would require further improvements in detection efficiency.

Escherichia coli transcription termination factor rho has a two-domain structure in its activated form.

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.

Isozyme phenotypes of polyoma virus tumors in mice.

Isozyme profiles for 32 enzyme systems were studied in tumors induced by two strains of polyoma virus (2PTA and LID1), in two conventional mouse strains (C3H/BiDa and NIH), and in athymic (nude) mice of two genetic backgrounds (C3H/Hes nu/nu and NIH nu/nu). Tumors studied were: primary and transplant passages of salivary gland tumors (127); primary thymic epithelial tumors (12); primary subcutaneous sarcomas (6); primary hair follicle tumors (5); primary and transplant passages of mammary tumors (18); primary ameloblastomas (3); and primary renal medullary sarcomas (3). Regardless of mouse strain or virus strain, the isozyme arrays were highly constant and unique for each tumor histotype with the exception of salivary and mammary tumors, which shared a single profile differing from that of each of the other histotype-associated profiles. Other tumor types could be distinguished from each other and from the salivary-mammary tumor pair by as few as five isozymes: glycerol-3-phosphate dehydrogenase; glyceraldehydephosphate dehydrogenase; lactate dehydrogenase; sorbitol dehydrogenase; and alkaline phosphatase. Twelve nonpolyoma mammary tumors and their passages from mouse mammary tumor virus-expressed C3H/Hes nu/+ mice were analyzed for the same enzymes; variations in activity and isozyme profiles were found for ten enzyme systems. Three spontaneous salivary myoepitheliomas in BALB/c mice were also analyzed; two different lactate dehydrogenase profiles were observed, and all three tumors lacked the placental alkaline phosphatase present in polyoma virus-induced salivary tumors. Uniformity of isozyme phenotype may be characteristic of DNA virus transformation of cells in a particular differentiative state. This uniformity does not appear to occur in mouse mammary tumor virus-associated tumors, spontaneous tumors, and, according to the literature, chemically induced tumors.

Calibration of a 238Pu,Be facility for partial-body measurements of organ cadmium.

An improved instrument is described for the measurement of liver and kidney cadmium by in vivo neutron activation analysis in both occupationally and environmentally exposed persons. Detailed calibrations of the instrument used in a study of 83 male workers at a cadmium production plant are give. The importance of accurate organ localisation by ultrasound is stressed, without which errors of 40 and 25% in individual and group kidney measurements, respectively, can occur. The detection limit (2 SD of the background) is 2.2 mg cadmium in the kidney and 1.5 micrograms g-1 (wet weight) in the liver for a local dose of 4.7 mSv. This instrument therefore combines the advantages of portability with high sensitivity of detection of cadmium.

Renal cadmium overload without nephrotoxicity.

A redundant nickel/cadmium battery worker was investigated for non-specific fatigue after completing five years in the industry. Sensitive techniques for in-vivo organ cadmium measurement showed a moderate accumulation in the liver but a very large concentration in the kidneys. Despite this, overall glomerular and tubular function were not impaired. It was concluded that the mechanism of proteinuria observed in some cadmium workers is obscure and not clearly related to the degree of kidney saturation with cadmium.

Critical concentrations of cadmium in human renal cortex: dose-effect studies in cadmium smelter workers.

Cadmium was measured in vivo in the left kidney and liver of 82 industrially exposed workers and 10 control subjects. The range of Cd values for the industrial group was 0.9-57 mg for the whole kidney and 0.8-120 ppm for the liver, compared to 0.4-11.8 mg and 0.6-7.9 ppm for the control group. Below 40 ppm in the liver, the kidney Cd burden tended to increase with increasing liver concentration. Above 40 ppm, the kidney Cd content decreased as the liver concentration increased. This biphasic relation between Cd in the kidney and the liver for all subjects showed a critical level of approximately 31 mg Cd in the kidney. Estimates of the critical level by beta 2-microglobulin and urinary protein measurements yielded critical values of 31-42 mg Cd for the whole kidney (300-400 microgram/g for the renal cortex).