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Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing., CZI NIH grant ERC grant (Thdefine)

The transcription factor E2A plays a crucial role in B cell development, the control of immunoglobulin gene rearrangement and expression. Here we report that in primary mouse B cells lipopolysaccharide (LPS) is able to induce the level of E2A protein by over 50-fold in days of culture. In contrast, CD40 signaling is insufficient to cause an E2A increase and can in fact prevent the LPS-mediated induction of E2A. These results suggest that E2A induction requires both proliferation and differentiation. We find that E2A protein induction is regulated post-transcriptionally since E2A mRNA is not induced by LPS. We have thus identified an important additional layer of regulation affecting the activity of E2A transcription factors.

Patients with end-stage renal disease (ESRD) may have quality-of-life (QOL) concerns that are not fully appreciated by their providers. The authors conducted focus groups with dialysis patients and dialysis professionals to determine whether this qualitative method would reveal differences between patients' and providers' views about: 1) domains of QOL that are affected by ESRD and dialysis; and 2) aspects of dialysis that affect QOL.Separate focus group discussions were held with: 8 adult hemodialysis patients (mean age 50 years; 3 women; mean duration of dialysis 8.5 years), 5 adult peritoneal dialysis patients (mean age 54 years; 3 women; mean duration of dialysis 4.6 years), 8 nephrologists (mean of 12 years of dialysis practice), and 9 other health professionals involved in dialysis care (3 nurses, 2 dietitians, 2 social workers, and 2 technicians; mean of 10 years experience in dialysis care). Discussions were audiotaped, transcribed verbatim, and reviewed independently by three investigators to identify and categorize distinct thoughts.1,271 distinct thoughts were identified and grouped into 20 related categories, which included ten QOL domains and ten aspects of dialysis that affect QOL. Compared with the professionals, the patients identified one additional relevant QOL domain (10 vs 9), and one additional aspect of dialysis that affects QOL (10 vs 9), and expressed more thoughts per domain (p0.05), although the contents of their comments were frequently similar. Among QOL domains, the numbers of related thoughts identified by patients and professionals, respectively, were: freedom/control (60, 89); social relationships (36, 11); anxiety (37, 4); role function (24, 10); energy (12, 10); body image (16, 4); sex (11, 21); mental attitude (21, 0); sleep (15, 1), and cognitive function (13, 7). Among aspects of dialysis that affect QOL, the numbers of thoughts identified by patients and professionals were: general dialysis issues (159, 105); relationships with staff (62, 110); patient education (63, 68); diet (44, 40); scheduling (57, 3); vascular or peritoneal access issues (31, 17), adaptation to dialysis (16, 14); dialysis dose (18, 8); symptoms (25, 0), and self-care (5, 24).Although health professionals have a good understanding of patient concerns about the effects of ESRD and dialysis, the focus group discussions revealed a breadth and depth of QOL concerns that they may not fully appreciate.

Upon treatment with lipopolysaccharide (LPS), primary B cells proliferate and differentiate into plasma cells with concomitant up-regulation of immunoglobulin (Ig) gene expression. Here we examine the role of the Igkappa 3' enhancer in this process using a kappa3'-enhancer-driven beta-globin reporter gene in transgenic mice. We find that LPS treatment up-regulates kappa3' enhancer activity as a function of differentiation rather than proliferation, since proliferation only (induced by cross-linking of CD40) is insufficient to activate the element, whilst differentiation with only limited proliferation (LPS + transforming growth factor-beta) does allow activation to occur. The Igkappa 3' enhancer is also induced by cross-linking of surface Ig and this signal can synergize with LPS activation, suggesting that distinct activation pathways are used. Nevertheless, both of these pathways can be inhibited by co-cross-linking of CD40. Thus Ig enhancers in the heavy and light chain loci are differentially regulated in response to CD40.

Hypertension may play an important role in the pathogenesis of the excess cardiovascular and cerebrovascular (CV) morbidity observed in hemodialysis patients (HD). However, the optimal blood pressure (BP) range for HD patients has not been defined. We postulated that there is a "U" curve relationship between BP and CV mortality. To explore this hypothesis we studied 5,433 HD patients in Dialysis Clinic Inc., a large not-for-profit chain, over a five year period.Cox regression, with fixed and time-varying covariates, was used to assess the effect of systolic blood pressure (SBP) and diastolic blood pressure (DBP), pre- and post-dialysis, on CV mortality, while adjusting for age, gender, ethnicity, primary cause of end-stage renal disease, Kt/V, serum albumin, and antihypertensive medications.The overall impact of BP on CV mortality was modest. Pre-dialysis, neither systolic nor diastolic hypertension were associated with an increase in CV mortality. Post-dialysis, SBPor = 180 mm Hg (RR = 1.96, P0.015) and DBPor = 90 mm Hg (RR = 1.73, P0.05) were associated with increased CV mortality. Low SBP (SBP110 mm Hg) was associated with increased CV mortality, pre- and post-dialysis.The results suggest the presence of a "U" curve relationship between SBP post-dialysis and CV mortality in HD patients.

The Ig kappa 3' enhancer is required for high levels of Ig kappa gene expression. We now show that kappa 3' enhancer function increases five- to eightfold after stimulation of primary murine B cells with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. In the presence of cyclosporin A this induction is almost halved, suggesting that transcription factors of the NFAT family contribute to kappa 3' enhancer induction. Indeed, we identify a novel NFAT binding site which is required for full enhancer function. We find that this site is transcriptionally active in stimulated B cells, T cells and fibroblasts and that both PMA and ionomycin are required for maximal induction. Time course analysis of the components of the protein-DNA complex in primary lymphocytes reveals that both NFATp and NFATc are present in the complex after 15 min, while only NFATc is detectable after 4 h. This suggests that NFATc plays the dominant role in controlling long-term responses of this transcription factor family. Furthermore, JunB, JunD, FosB and cFos form part of the DNA-protein complex in Bal-17 B cells. Complex formation as well as transcriptional activity can also be induced by crosslinking of surface Ig. We have, thus, identified a unique NFAT complex in B cells that contributes to Ig kappa gene expression.

ESRD Network Number 1, composed of Maine, New Hampshire, Vermont, Massachusetts, Connecticut, and Rhode Island, developed a Network Core Indicator Pilot Project using the dialysis units represented on the Medical Review Board. Network 1's Core Indicator Pilot Project aimed to (1) estimate the proportion of end-stage renal disease (ESRD) patients in Network 1 receiving hemodialysis treatments associated with a urea reduction ratio less than 60% to 65%, (2) elucidate the patient characteristics associated with a hemodialysis dose less than 65%, (3) define the processes in the delivery of hemodialysis that limit the provision of an adequate dialysis dose, and (4) initiate the routine collection of measures of dialysis dose, the analysis of those data, and feedback to the participating dialysis units. In the course of the Core Indicator Pilot Project, we observed little uniformity in the sampling method for the postdialysis blood urea nitrogen sample. Thirty-three percent of the hemodialysis units reported that this critical blood sample was drawn immediately before the dialysis treatment was terminated; 25% were obtained immediately at the end of the dialysis treatment and 42% drew the sampleor = 5 minutes after all blood was reinfused to the patient. Especially in the presence of unappreciated blood recirculation in the angioaccess or postdialysis urea rebound, the lack of standardization in obtaining this critical blood sample to support the urea reduction ratio calculation greatly compromises any comparisons of performance across dialysis facilities and may jeopardize patient care. Future ESRD quality improvement efforts must focus not only on the results of the outcome measure but also on the process by which the measure is achieved. These fundamental principles of quality assessment should be considered by policy specialists, payers, providers, and developers of clinical practice guidelines.

Benzene is a ubiquitous occupational hematotoxin and leukemogen, but people vary in their response to this toxic agent. To evaluate the impact of interindividual variation in enzymes that activate (i.e., CYP2E1) and detoxify (i.e., NQO1) benzene and its metabolites, we carried out a case-control study in Shanghai, China, of occupational benzene poisoning (BP; i.e., hematotoxicity), which we show is itself strongly associated with subsequent development of acute nonlymphocytic leukemia and the related myelodysplastic syndromes (relative risk, 70.6; 95% confidence interval, 11.4-439.3). CYP2E1 and NQO1 genotypes were determined by PCR-RFLP, and CYP2E1 enzymatic activity was estimated by the fractional excretion of chlorzoxazone (fe(6-OH)) for 50 cases of BP and 50 controls. Subjects with both a rapid fe(6-OH). and two copies of the NQO1 609C--T mutation had a 7.6-fold (95% confidence interval, 1.8-31.2) increased risk of BP compared to subjects with a slow fe(6-OH) who carried one or two wild-type NQO1 alleles. In contrast, the CYP2E1 PstI/RsaI polymorphism did not influence BP risk. This is the first report that provides evidence of human susceptibility to benzene-related disease. Further evaluation of susceptibility for hematotoxicity and hematological malignancy among workers with a history of occupational exposure to benzene is warranted.

The expression of naked plasmid DNA coding for firefly luciferase (pRSVluc) or a secreted protein, human-alpha-1-antitrypsin (pRcCMVhAAT) in mouse skeletal muscle was characterized following administration by an improved intramuscular injection technique. Injection guided by intense illumination along the longitudinal axis of the mouse quadriceps muscle and parallel to the myofibers yielded 200-fold higher levels of luciferase expression than perpendicular injection. Luciferase expression was inhibited by an excess of non-coding DNA or dextran sulfate suggesting that muscle DNA uptake mechanism(s) can be saturated. Injected plasmid DNA was rapidly eliminated from the muscle as evidenced by tissue distribution studies of radiolabeled hAAT plasmid and Southern analysis. However, PCR analysis demonstrated that hAAT cDNA persisted in the muscle for at least 1 month after injection. Immunohistochemistry techniques indicated that the hAAT gene was expressed by the muscle fibers. ELISA analysis of serum samples collected from intramuscularly injected mice demonstrated that secreted hAAT protein concentration peaked in serum by day 7, started to decline by day 14 and was barely detectable 21 days post-injection. RT-PCR analysis demonstrated that hAAT transcript persisted at the site of injection for at least 1 month indicating that the decline of serum hAAT concentration 21 days post-injection was not due to the absence of hAAT transcript. However, the decline of hAAT protein concentration in the serum was inversely correlated with accumulation of murine anti-hAAT antibodies in circulation.

Intratracheal administration of plasmid DNA resulted in gene expression in mouse airways in the absence of any enhancing agent. Administration of plasmid DNA encoding the chloramphenicol acetyltransferase gene (CAT) in sterile water lead to CAT transgene expression that peaked between 1 and 3 days and was detected up to 28 days after DNA administration. Transgene expression was independent of mouse gender, age and strain. Levels of expression from DNA in various isotonic solutions did not differ from levels obtained with DNA administered in water, suggesting that transfection is not dependent on damage to airway cells caused by a hypo-osmotic delivery vehicle. Pharmacokinetic studies using radiolabeled plasmid DNA showed that DNA was rapidly degraded, while higher levels of radioactivity were retained for longer duration following administration of cationic liposome-DNA complexes in the airway. Southern blot and PCR analysis confirmed that DNA complexed with DOTMA-DOPE was retained in the airways for a longer period. However, cationic liposomes DOTMA-DOPE (1:1) or DOTAP complexed with DNA, did not enhance expression over DNA alone. These results suggest that 'naked' plasmid DNA should be included as a control in all studies on intratracheal gene delivery using nonviral systems.

Cisplatin treatment causes tubulointerstitial injury to the kidneys. Common clinical syndromes associated with its use include acute renal failure and a magnesium wasting state. Routine fluid infusion therapy has markedly reduced the incidence of acute renal failure. However, methodologic limitations of most recent studies preclude confident conclusions regarding long-term effects of cisplatin treatment on renal function. Experimental and clinical evidence suggests that sulfhydryl metabolism and oxidative stress are central to cisplatin renal injury. Both in animals and humans, a variety of agents appear to alter renal uptale of cisplatin and its hemodynamic consequences, thus modulating nephrotoxicity. Thiosulfates and related agents have received most study. Other agents examined have included calcium channel blockers, bismuth, selenium, glycine, cimetidine, and probenecid.

Benzene, an important industrial chemical, is myelotoxic and leukemogenic in humans. It is metabolized by cytochrome P450 2E1 to various phenolic metabolites which accumulate in the bone marrow. Bone marrow contains high levels of myeloperoxidase which can catalyze the further metabolism of the phenolic metabolites to reactive free radical species. Redox cycling of these free radical species produces active oxygen. This active oxygen may damage cellular DNA (known as oxidative DNA damage) and induce genotoxic effects. Here we report the induction of oxidative DNA damage by benzene and its phenolic metabolites in HL60 cells in vitro and in the bone marrow of C57BL/6 x C3H F1 mice in vivo utilizing 8-hydroxy-2'-deoxyguanosine as a marker. HL60 cells (a human leukemia cell line) contain high levels of myeloperoxidase and were used as an in vitro model system. Exposure of these cells to phenol, hydroquinone, and 1,2,4-benzenetriol resulted in an increased level of oxidative DNA damage. An increase in oxidative DNA damage was also observed in the mouse bone marrow in vivo 1 h after benzene administration. A dose of 200 mg/kg benzene produced a 5-fold increase in the 8-hydroxydeoxyguanosine level. Combinations of phenol, catechol, and hydroquinone also resulted in significant increases in steady state levels of oxidative DNA damage in the mouse bone marrow but were not effective when administered individually. Administration of 1,2,4-benzenetriol alone did, however, result in a significant increase in oxidative DNA damage. This represents the first direct demonstration of active oxygen production by benzene and its phenolic metabolites in vivo. The conversion of benzene to phenolic metabolites and the subsequent production of oxidative DNA damage may therefore play a role in the benzene-induced genotoxicity, myelotoxicity, and leukemia.

Adjuvant therapy with ablative radioiodine after surgical resection of apparently localized thyroid carcinoma remains controversial because of the favorable prognosis of thyroid carcinoma and the risk of leukemia from the radioiodine. No controlled trials have been performed to examine this issue. We constructed a decision analytic model to examine whether patients with apparently localized thyroid carcinoma should receive radioiodine. Our analysis suggests that radioiodine modestly improves life expectancy by 2 to 15 months, depending on the patient's age and sex. This model predicts that the benefit of a reduction in the likelihood of recurrence outweighs the risk of leukemia from radioiodine.

Physicians and other health care workers are concerned about their occupational risk of acquiring human immunodeficiency virus infection. We have developed an approach that can help health care workers estimate their cumulative risk of infection with human immunodeficiency virus. Illustrations are used to develop a score that reflects an individual's occupational exposures and social behavior. This score is then translated into the probability that the worker is infected.

The effects of mesothermic anaerobic or aerobic sludge digestion on survival of eggs from the roundworms Ascaris suum, toxocara canis, Trichuris vulpis, and Trichuris suis and from the rat tapeworm Hymenolepis diminuta were studied. Destruction of eggs throughout a 15-day treatment period, as well as their viabilities after reisolation, was analyzed. The laboratory model digesters used in this study were maintained at a 15-day retention schedule, partially simulating a continuously operating system. Ascaris eggs were destroyed in the anaerobic (23%) or aerobic (38%) digesters, and 11% Trichuris eggs were destroyed in the aerobic digesters. Trichuris eggs in anaerobic digesters and Toxocara eggs in either anaerobic or aerobic digesters were not destroyed. Destruction of eggs in digesters was correlated with the state of the eggs before subjection to the treatment processes; i.e., some Ascaris and Trichuris eggs were already embryonated in host intestinal contents or feces and hence past their most resistant stage. The viabilities of Ascaris and Toxocara eggs that survived the digestion processes were greater in anaerobically treated than in aerobically treated material. Eggs from Hymenolepis were nonviable before use in the experiments. However, they were more effectively destroyed in aerobic digesters than in anaerobic digesters.

Destruction rates of parasite eggs in stored sludge were examined to help understand the fate of these agents of enteric diseases in sludge lagoons. Eggs from the roundworms, Ascaris spp., Toxocara spp., Trichuris spp., and the tapeworm, Hymenolepis spp., were treated with domestic sludges by aerobic or anaerobic processes. Sludge samples seeded with eggs were stored at 4 or 25 degrees C or in a container inserted into the ground to simulate lagoon conditions. The number of eggs recovered from the samples decreased with storage time. The viability and infectivity of eggs recovered were related to the storage temperature; i.e., the eggs stored at 4 degrees C remained viable longer than those stored at 25 degrees C. After 25 months at 4 degrees C, the Toxocara eggs and some Ascaris eggs remained both viable and infective, whereas most of these eggs stored at 25 degrees C were rendered nonviable after 10 to 16 months of storage in sludge. Although storage temperature was found to be the most important factor affecting the destruction and viability of these eggs, other factors, such as the type of sludge digestion, whether or not the eggs were digested along with the sludge or added later, storage in the soil versus sludge, pH, and egg species also exhibited some minor effects. These controlled laboratory studies suggest that lagooning of sludge can be an effective method for the elimination of parasite eggs, particularly in warmer geographical locations.

Seventy-eight newborn pigs were allotted to 4 treatment groups: 22 pigs in group A were given no selenium-vitamin E (Se-E), 22 pigs in group B were given small doses of Se-E, 22 pigs in group C were given medium doses of Se-E, and 12 pigs in group D were given large doses of Se-E. Pigs were intramuscularly injected before 7 days of age and at weaning (40 days of age), respectively, as follows: group A--1 ml of physiologic saline solution/pig each time, group B--0.25 mg of Se/pig and later 0.06 mg of Se/kg of body weight, group C--1.0 mg of Se/pig and later 0.24 mg of Se/kg, and group D--1.5 mg of Se/pig and later 0.72 mg of Se/kg. Selenium was supplied as sodium selenite in commercially available Se-E injectable compounds. From 2 weeks of age to weaning, the pigs were fed a corn-torula yeast creep feed containing Se at the concentration of 0.03 ppm, and from weaning to slaughter, a corn-soybean meal ration was fed containing Se at the concentration of 0.07 ppm and alpha-tocopherol at the concentration of 15.7 mg/kg. Subclinical Se-E deficiency developed in control pigs of group A and was characterized by subtle muscular stiffness, significant increases in plasma activities of glutamic oxalacetic transaminase (GOT) and creatine phosphokinase (CPK), and typical residual lesions in heart and skeletal muscle, but not in liver, at slaughter at 165 days of age. Pigs injected with Se-E did not develop these evidences of subclinical deficiency. Pigs in group D were stunted for several weeks after the 2nd Se-E injection, and plasma GOT and CPK activities were significantly increased at 3 weeks after injection. Growth rates were otherwise similar between groups. Significant difference in Se content of liver, muscle, serum, and hair was not seen between pigs in the 4 groups at 120 and 165 days of age. A test period of physical exertion and heat stress resulted in significant increase of plasma GOT and CPK activities in 4 of 8 pigs at 110 days of age.

The authors examine data on the risk of human immunodeficiency virus (HIV) infection in various low- and high-risk populations and in surgical personnel. They conclude that the risk of HIV infection to surgeons, nurses, and technicians using barrier precautions is of the same order of magnitude as the risk to sexual participants who use barrier precautions. Further, the authors assert that routine preoperative screening could be even more socially destructive than screening the general population because privacy will be more seriously compromised and because the occurrence of false-positive tests is probably higher in chronically ill patients than in healthy persons. They question whether it is rational or ethical to imply that preventing HIV infection in surgical personnel is more important than preventing such infection in others.

Two glyceryl ethers, 1-O-hexadecyl glycerol and 1-O-cis-octadec-11-enyl glycerol, chimyl and paramecyl alcohol, respectively, were quantified in total phospholipids and five glycerophospholipid classes from cells and cilia of the ciliated protozoon, Paramecium tetraurelia. The ether content of 2-aminoethyl phosphonoglycerolipid was 85-90 mole %. Concentrations of ethers were greatest in the ethanolamine phosphonolipids greater than phosphatidylcholines greater than phosphatidylserines greater than phosphatidylethanolamines greater than phosphatidylinositols. The glyceryl ether concentrations in total cellular phospholipids increased with culture age in P. tetraurelia and P. multimicronucleatum cells. The glyceryl ether concentrations in the phospholipids of P. tetraurelia cilia remained constant from mid log to stationary phase of culture growth. Paramecium tetraurelia phospholipid glyceryl ether concentrations were made greater by supplementation of cultures with chimyl alcohol.