Peppered with lead: An environmental forensics approach to identify the source of rising blood lead levels.

Despite the phase-out of lead-based products, lead contamination can still present a contemporary risk to public health. In situations where elevated blood lead cannot be attributed to common sources, detailed environmental investigation is needed to identify more elusive sources and manage harmful exposure pathways. We apply a forensics approach to assess common and elusive sources of lead in the home environment of two individuals with fluctuating blood lead levels in Sydney, Australia. Using multiple analytical lines of evidence (portable X-Ray Fluorescence spectrometry (pXRF), inductively coupled-plasma mass spectrometry (ICP-MS), lead isotopic compositional analysis (PbIC) and haematological assessment) a pewter pepper grinder containing lead (>6000 mg/kg; 70% bioavailable) was identified as a potential source. After removing the pepper grinder from the home, the couple's blood lead decreased to below the Australian intervention level of 5 µg/dL within a year (Person A: from 12.5 µg/dL in August 2020 to 4.4 µg/dL in March 2022; and Person B: 15.4 µg/dL in August 2020 to 2.1 µg/dL in July 2021). This case study demonstrates how environmental science investigations can play a crucial role in supporting people to take evidence-based action to improve their health.

Selective Chelation of the Exotic Meitner-Auger Emitter Mercury-197 m/g with Sulfur-Rich Macrocyclic Ligands: Towards the Future of Theranostic Radiopharmaceuticals.

Mercury-197 m/g are a promising pair of radioactive isomers for incorporation into a theranostic as they can be used as a diagnostic agent using SPECT imaging and a therapeutic via Meitner-Auger electron emissions. However, the current absence of ligands able to stably coordinate 197m/g Hg to a tumour-targeting vector precludes their use in vivo. To address this, we report herein a series of sulfur-rich chelators capable of incorporating 197m/g Hg into a radiopharmaceutical. 1,4,7,10-Tetrathia-13-azacyclopentadecane (NS4 ) and its derivatives, (2-(1,4,7,10-tetrathia-13-azacyclopentadecan-13-yl)acetic acid (NS4 -CA) and N-benzyl-2-(1,4,7,10-tetrathia-13-azacyclopentadecan-13-yl)acetamide (NS4 -BA), were designed, synthesized and analyzed for their ability to coordinate Hg2+ through a combination of theoretical (DFT) and experimental coordination chemistry studies (NMR and mass spectrometry) as well as 197m/g Hg radiolabeling studies and in vitro stability assays. The development of stable ligands for 197m/g Hg reported herein is extremely impactful as it would enable their use for in vivo imaging and therapy, leading to personalized treatments for cancer.

Trace element contamination of soil and dust by a New Caledonian ferronickel smelter: Dispersal, enrichment, and human health risk.

Metallurgical industries remain a considerable source of trace element contamination and potential human health risk. Determination of sources is a key challenge. With respect to the South Pacific's largest and longest operating metallurgic smelter in Nouméa, New Caledonia, determining the environmental impact and subsequent human health risk associated with local ferronickel smelting is complicated by natural geological enrichment of Ni and Cr. This study applies a multi-method and multi-matrix approach to disentangle smelter emissions from geogenic sources and model the consequent health risk from industrial activity. Dust wipes (n = 108), roadside soil (n = 91), garden soil (n = 15) and household vacuum dust (n = 39) were assessed to explore geospatial trace element (As, Cr, Cu, Fe, Mn, Ni, Pb, S, V and Zn) variations across outdoor and indoor environments. Enrichment factors (EF) identified elevated levels of smelter-related trace elements: S (EF = 7), Ni (EF = 6) and Cr (EF = 4), as well as Zn (EF = 4). Smelter-related elements in soil and dust deposits were negatively correlated with distance from the facility. Similarity of Pb isotopic compositions between dust wipes, surface soil and vacuum dust indicated that potentially toxic trace elements are being tracked into homes. Non-carcinogenic health risk modelling (Hazard Index, HI) based on 15 spatial nodes across Nouméa revealed widespread exceedance of tolerable risk for children (0-2 years) for Ni (HI 1.3-15.8) and Mn (HI 0.6-1.8). Risk was greatest near the smelter and to the north-west, in the direction of prevailing wind. Given the elevated cancer risk documented in New Caledonia, disentanglement of environmental from industrial sources warrants further attention to ensure community health protection. Our analysis illustrates how the confounding effects from complex environmental factors can be distilled to improve the accuracy of point source apportionment to direct future mitigation strategies.

Clinical utility of skin cancer and melanoma risk scores for population screening: TRoPICS study.

BACKGROUND: Screening for skin cancer can be cost-effective if focused on high-risk groups. Risk prediction tools have been developed for keratinocyte cancers and melanoma to optimize advice and management. However, few have been validated in a clinical setting over the past few years. OBJECTIVES: To assess the clinical utility of risk assessment tools to identify individuals with prevalent skin cancers in a volunteer-based screening clinic. METHODS: Participants were adults presenting for a skin check at a volunteer-based skin cancer screening facility. We used previously published tools, based on questionnaire responses, to predict melanoma and keratinocyte cancers [KCs; basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)] and classified each participant into one of five risk categories. Participants subsequently underwent a full skin examination by a dermatologist. All suspicious lesions were biopsied, and all cancers were histopathologically confirmed. RESULTS: Of 789 people who presented to the clinic, 507 (64%) consented to the study. Twenty-two BCCs, 19 SCCs and eight melanomas were diagnosed. The proportion of keratinocyte cancers diagnosed increased according to risk category from <1% in the lowest to 24% in the highest risk category (P < 0.001). Subtype analysis revealed similar proportionate increases in BCC or SCC prevalence according to risk category. However, a similar proportion of melanoma cases were detected in the low-risk and high-risk groups. CONCLUSION: The risk prediction model for keratinocyte cancers can reliably identify individuals with a significant skin cancer burden prior to a skin examination in the community setting. The prediction tool for melanoma needs to be tested in a larger sample exposed to a wider range of environmental risk factors.

Measuring genetic knowledge: a brief survey instrument for adolescents and adults.

Basic knowledge of genetics is essential for understanding genetic testing and counseling. The lack of a written, English language, validated, published measure has limited our ability to evaluate genetic knowledge of patients and families. Here, we begin the psychometric analysis of a true/false genetic knowledge measure. The 18-item measure was completed by parents of children with congenital heart defects (CHD) (n = 465) and adolescents and young adults with CHD (age: 15-25, n = 196) with a mean total correct score of 12.6 [standard deviation (SD) = 3.5, range: 0-18]. Utilizing exploratory factor analysis, we determined that one to three correlated factors, or abilities, were captured by our measure. Through confirmatory factor analysis, we determined that the two factor model was the best fit. Although it was necessary to remove two items, the remaining items exhibited adequate psychometric properties in a multidimensional item response theory analysis. Scores for each factor were computed, and a sum-score conversion table was derived. We conclude that this genetic knowledge measure discriminates best at low knowledge levels and is therefore well suited to determine a minimum adequate amount of genetic knowledge. However, further reliability testing and validation in diverse research and clinical settings is needed.

The impact of osteoblastic differentiation on osteosarcomagenesis in the mouse.

Osteosarcomas remain an enigmatic group of malignancies that share in common the presence of transformed cells producing osteoid matrix, even if these cells comprise a minority of the tumor volume. The differentiation state of osteosarcomas has therefore become a topic of interest and challenge to those who study this disease. In order to test how the cell of origin contributes to the final state of differentiation in the transformed cells, we compared the relative tumorigenicity of Cre-LoxP conditional disruption of the cell cycle checkpoint tumor-suppressor genes Trp53 and Rb1 using Prx1-Cre, Collagen-1α1-Cre and Osteocalcin-Cre to transform undifferentiated mesenchyme, preosteoblasts and mature osteoblasts, respectively. The Prx1 and Col1α1 lineages developed tumors with nearly complete penetrance, as anticipated. Osteosarcomas also developed in 44% of Oc-Cre;Rb1(fl/fl);Trp53(fl/fl) mice. We confirmed using 5-ethynyl-2'-deoxyuridine click chemistry that the Oc-Cre lineage includes very few actively cycling cells. By assessing radiographic mineralization and histological osteoid production, the differentiation state of tumors did not correlate with the differentiation state of the lineage of origin. Some of the osteocalcin-lineage-derived osteosarcomas were among the least osteoblastic. Osteocalcin immunohistochemistry in tumors correlated well with the expression of DNA methyl transferases, suggesting that silencing of these epigenetic regulators may influence the final differentiation state of an osteosarcoma. Transformation of differentiated, minimally proliferative osteoblasts is possible but may require such an epigenetic reprogramming that the tumors no longer resemble their differentiated origins.

Longitudinal expression of type I interferon responsive genes in systemic lupus erythematosus.

Cross-sectional studies of patients with systemic lupus erythematosus (SLE) have demonstrated an association between activation of type I interferon (IFN) pathway and disease activity. This study examined longitudinal changes in IFN-regulated gene expression in peripheral blood using microarrays. A cross-section of 66 patients from the Autoimmune Biomarkers Collaborative Network SLE archive was evaluated. We also examined paired samples from a 15 patient subset collected during a period of low disease activity (Baseline) and at a subsequent flare event, and baseline scores of 29 patients who maintained low disease activity. IFN response (IFNr) scores were calculated from three IFN-regulated genes. Overall, higher IFNr scores were associated with increased disease activity. However, IFNr scores were not significantly different between the paired Baseline and Flare samples. An extended longitudinal analysis in 11 patients indicated little change in IFNr scores over time, even during dynamic disease activity. In patients with low disease activity, IFNr scores were not different between patients who experienced a subsequent flare and those who maintained low disease activity. In summary, although higher IFNr scores were associated with greater disease activity, IFNr scores of individual patients did not correlate with changes in disease severity or flare risk.

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques.

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with Mr of approximately 30,000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30,000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography-mass spectrometry, and found to consist of two related species of approximately 28,100 and 26,500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.

Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by "proteome" technologies.

BACKGROUND: There is great interest in characterizing the proteins of the gastric pathogen, Helicobacter pylori, especially those proteins to which humans respond immunologically. Such proteins have potential importance in diagnosis and vaccine development. METHODS: Two-dimensional gel electrophoresis in combination with Western blotting was used to separate and identify potential antigens of Helicobacter pylori strain Z-170. Proteins found to be reactive with pooled sera from 14 infected patients were individually digested in situ with endoproteinase Lys-C, and the resulting fragments were analyzed by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Over 20 proteins were reactive in Western blots with pooled sera from 14 infected patients. The mass spectral data was compared with predictions from the H. pylori genome DNA sequence. Each of the 20 proteins was readily identified. CONCLUSIONS: We propose that this "proteome" approach for identification of previously unknown proteins will be useful in examining regulation of H. pylori gene expression and protein localization in the development of improved serologic tests to detect and monitor H. pylori infection. This approach will also be useful for identifying potential targets for antimicrobial or vaccine development for H. pylori and other pathogens whose genomes have been sequenced.

Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by two-dimensional gel electrophoresis, sequence analysis, and serum profiling.

There is great interest in characterizing the proteins of the gastric pathogen Helicobacter pylori, especially those to which humans respond immunologically, because of the potential importance of such proteins in diagnosis and vaccine development. Two-dimensional gel electrophoresis was used to separate and identify potential antigens of H. pylori ATCC 43504. Over 30 proteins were reactive in Western blots with pooled sera from 14 infected patients. These proteins were analyzed by N-terminal sequence analysis. Fourteen proteins were determined to be distinct from any proteins previously described from H. pylori; the others were previously isolated and characterized proteins. Analysis of eight distinct H. pylori strains showed that most of these antigens were produced by all of the strains. We propose that collection of new antigens such as those recognized here will be useful in serologic tests for detecting and monitoring H. pylori infection and may also serve as potential targets for antimicrobial agent or vaccine development.

Sequence variation and phylogenetic analysis of envelope glycoprotein of hepatitis G virus.

A transfusion-transmissible agent provisionally designated hepatitis G virus (HGV) was recently identified. In this study, we examined the variability of the HGV genome by analysing sequences in the putative envelope region from 72 isolates obtained from diverse geographical sources. The 1561 nucleotide sequence of the E1/E2/NS2a region of HGV was determined from 12 isolates, and compared with three published sequences. The most variability was observed in 400 nucleotides at the N terminus of E2. We next analysed this 400 nucleotide envelope variable region (EV) from an additional 60 HGV isolates. This sequence varied considerably among the 75 isolates, with overall identity ranging from 79.3% to 99.5% at the nucleotide level, and from 83.5% to 100% at the amino acid level. However, hypervariable regions were not identified. Phylogenetic analyses indicated that the 75 HGV isolates belong to a single genotype. A single-tier distribution of evolutionary distances was observed among the 15 E1/E2/NS2a sequences and the 75 EV sequences. In contrast, 11 isolates of HCV were analysed and showed a three-tiered distribution, representing genotypes, subtypes, and isolates. The 75 isolates of HGV fell into four clusters on the phylogenetic tree. Tight geographical clustering was observed among the HGV isolates from Japan and Korea.

Sequence variation and phylogenetic analysis of the 5' terminus of hepatitis G virus.

We determined the nucleotide and deduced amino acid sequence of the 5' terminus of the hepatitis G virus (HGV) genome from isolates of varied geographical origins. Our analysis showed that the putative 5' non-coding region (NCR) contains several blocks of highly conserved sequences that may be useful for the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of HGV RNA. Overall, the degree of conservation within the 669-nucleotide (nt) 5'terminal sequence was found to range from 99.5% to 86% sequence identity. We also showed that the HGV NCR from some isolates contained conserved insertions or deletions that altered the translational reading frames at the 5'-end of the genome, resulting in different sizes of predicted polyproteins encoded by genomes of individual isolates. Specifically, the insertions/deletions affected the size of the peptide preceding the putative first envelope (E1) protein. Phylogenetic analysis of the nucleotide sequences suggested that the isolates examined can be classified into distinct groups that may be useful for studying the molecular evolution of HGV and possible relationships between isolate sequence characteristics and infection patterns.

Molecular characterization of the hepatitis G virus.

A novel virus associated with acute and chronic hepatitis has been cloned and characterized. The single-stranded RNA genome is approximately 9000 nucleotides long and has the structure of a virus in the Flaviviridae family. The genes of the virus have been identified and characterized by a number of molecular techniques and a genetic map has been determined.

VH gene use by HIV type 1-associated lymphoproliferations.

The pathogenesis of polyclonal HIV-associated lymphomas lacking traditional B cell cofactors (i.e., Epstein-Barr virus [EBV] infection, c-myc translocations) is poorly understood. A multistep pathogenesis model has been proposed in which polyclonal lymphomas represent an earlier stage in HIV-associated lymphomagenesis before the emergence of a dominant malignant clone. Chronically present antigens have been proposed as a likely stimulus for polyclonal B cell proliferation; if so, polyclonal lymphoma-associated immunoglobulins (Igs) should have molecular evidence of somatic hypermutation, a process by which antibody affinity maturation in response to chronic antigenic stimulation occurs. Molecular analyses of Ig heavy chain variable (V(H)) gene use by B cells in a polyclonal HIV-associated large cell lymphoma lacking EBV and c-myc rearrangement was undertaken. Eighteen randomly selected clones generated from RT-PCR yielded 15 unique V(H) sequences, all of which were most homologous to only three previously identified germline V(H)1 genes. Two sets of clones (consisting of three and two clones, respectively) had identical V(H) gene sequences, and one pair of clones had identical third complementarity determining regions (CDR3s) but different V(H) gene sequences; eight clones were <95% homologous to their most related germline V(H)1 genes. We compared these results with Ig V(H)1 gene use by B cells present in a reactive hyperplastic lymph node obtained from an HIV-1-infected individual. Fifteen clones randomly selected from RT-PCRs yielded 15 unique V(H)1 sequences, all of which were most homologous to 5 previously identified germline V(H)1 genes; 10 clones were <95% homologous to their most related germline gene. Binomial probability analysis revealed that only 1 of the 15 unique V(H)1 sequences derived from the polyclonal lymphoma (i.e., 7%), as compared with 5 of 15 unique V(H)1 sequences derived from the reactive lymph node (i.e., 33%), had a low probability of occurrence by random chance (p < 0.05). These data provide molecular evidence of polyclonality in an HIV-associated polyclonal lymphoma, demonstrate a qualitative difference in somatic hypermutations of Ig V(H) genes associated with malignant versus reactive B cell lymphoproliferations, and support an antigen-mediated multistep pathogenesis model of HIV-1-associated lymphomagenesis.

Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent.

An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

Potency measurement of factor VIII in recombinant concentrates.

In preliminary studies on the stability of recombinant factor VIII (rFVIII) concentrates post reconstitution, a rise in potency to 200% of labelled values was observed in concentrates stored at 22 °C over 24 h. This was observed in potency estimates by the one-stage clotting, but not the two-stage clotting or chromogenic assays, and was not observed for intermediate-purity product derived from plasma (IPVIII). Use of human serum albumin (HSA), rather than the usual bovine material (BSA), to dilute product for the stability study abolished the rise in potency. Incorporating purified von Willebrand Factor (VWF) in the diluent buffer abolished the rise observed in BSA. A similar rise was observed upon incubating rFVIII in the presence of 10(-4) u of thrombin per mL in HSA buffer. Potency estimates using the HSA in the dilution buffer resulted in severe underpotency in relation to the label claim when using the two-stage clotting and the chromogenic assays, but not the one-stage clotting assay. Predilution in severe haemophilic plasma restored potency levels to those claimed. We conclude that (i) commercial preparations of BSA may be unsuitable for inclusion in buffers for rFVIII studies; (ii) FVIII in rFVIII concentrates is exquisitely sensitive to activation by thrombin, presumably as a result of the lack of VWF; (iii) accurate potency estimation in the two-stage assay systems requires VWF in the assay system.

Isolation and characterization of a chimpanzee monoclonal antibody to the G glycoprotein of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract disease in infants and young children. In this study a hybridoma line secreting a chimpanzee monoclonal antibody that neutralizes RSV was isolated. Two chimpanzees were immunized with recombinant vaccinia viruses that express the RSV F or G surface glycoprotein and 1 month later were infected intranasally with the wild-type RSV strain A2. Peripheral blood lymphocytes obtained from the animals were transformed with Epstein-Barr virus, and lymphoblastoid cell lines that secreted anti-RSV antibodies were identified by an RSV antigen-binding enzyme-linked immunosorbent assay. Supernatants from RSV antibody-secreting lymphoblastoid cell lines were tested for in vitro virus neutralization before being fused to the heteromyeloma cell GLI-H7. A chimpanzee antibody [immunoglobulin G3(lambda) subclass] produced from a hybridoma line designated E1.4/2 was shown to bind to the RSV G glycoprotein and neutralize a panel of subgroup A viruses, but not subgroup B viruses, at low (nanomolar) concentrations. Mice passively immunized with this antibody were partially resistant to RSV strain A2 challenge. The usefulness of such antibodies in immunoprophylaxis and immunotherapy of RSV infection is discussed.

Localization of antigenic sites on human cytomegalovirus virion structural proteins encoded by UL48 and UL56.

Two human cytomegalovirus (HCMV) virion structural proteins and their associated reading frames have been identified with two human-derived monoclonal antibodies (HMAbs), X2-16 and X-16. HMAb X2-16 identified recombinant protein expressing molecular clones that mapped to the open reading frame (ORF) of the UL48 gene of HCMV, between amino acids 584 and 646 (nucleotides 65,084 and 65,272, Chee et al., 1990, "Current Topics in Microbiology and Immunology," Vol. 154, pp. 125-169). The UL48 gene product has an apparent molecular weight of 216 kDa. HMAb X-16 identified clones derived from the UL56 ORF between amino acids 380 and 425 (nucleotides 84,733 and 84,870). On immunoblots, HMAb X-16 detected two HCMV proteins of 96 and 60 kDa. Both UL48 and UL56 are highly conserved among the human herpesviruses and their products have been predicted to have essential functions for virus production and maturation. These results confirm that UL48 and UL56 are functional genes encoding essential viral proteins which also generate an immune response in the immunocompetent host.

The production and characterisation of monoclonal antibodies to Lucilia cuprina larval antigens.

A panel of murine monoclonal antibodies was produced against three Lucilia cuprina larval preparations that are unlikely to be exposed to the sheep's immune system during a normal infection. Antibodies were successfully produced against a crude third instar midgut homogenate preparation (MG), and Sodium dodecyl sulphate (SDS) and Triton X-114 (TX) detergent extracts of first instar larvae. Characterisation of the relevant antigens was performed using 1- and 2D gel electrophoresis and immunoblotting, immunoperoxidase histological studies and in vitro larval growth cultures. All the mAbs were of the IgM isotype. Common recognition of bands at 40, 50 and 80 kDA was evident on 1D blots of larval organ preparations by most mAbs while recognition of antigens in the 2D blots appeared to be more specific. Immunohistological studies suggested that a number of the antibodies specifically bound to intracellular structures within the midgut epithelium. However, antibodies derived from one clone also recognised the epithelium of Malpighian tubules, oenocytes and muscle fibres. None of the antibodies raised against TX extracts were observed to bind to larval structures. Results of larval cultures suggested that certain antibodies could significantly inhibit larval survival and growth in vitro.