Motivation and context of concurrent stimulant and opioid use among persons who use drugs in the rural United States: a multi-site qualitative inquiry.

BACKGROUND: In recent years, stimulant use has increased among persons who use opioids in the rural U.S., leading to high rates of overdose and death. We sought to understand motivations and contexts for stimulant use among persons who use opioids in a large, geographically diverse sample of persons who use drugs (PWUD) in the rural settings. METHODS: We conducted semi-structured individual interviews with PWUD at 8 U.S. sites spanning 10 states and 65 counties. Content areas included general substance use, injection drug use, changes in drug use, and harm reduction practices. We used an iterative open-coding process to comprehensively itemize and categorize content shared by participants related to concurrent use. RESULTS: We interviewed 349 PWUD (64% male, mean age 36). Of those discussing current use of stimulants in the context of opioid use (n = 137, 39%), the stimulant most used was methamphetamine (78%) followed by cocaine/crack (26%). Motivations for co-use included: 1) change in drug markets and cost considerations; 2) recreational goals, e.g., seeking stronger effects after heightened opioid tolerance; 3) practical goals, such as a desire to balance or alleviate the effects of the other drug, including the use of stimulants to avoid/reverse opioid overdose, and/or control symptoms of opioid withdrawal; and 4) functional goals, such as being simultaneously energized and pain-free in order to remain productive for employment. CONCLUSION: In a rural U.S. cohort of PWUD, use of both stimulants and opioids was highly prevalent. Reasons for dual use found in the rural context compared to urban studies included changes in drug availability, functional/productivity goals, and the use of methamphetamine to offset opioid overdose. Education efforts and harm reduction services and treatment, such as access to naloxone, fentanyl test strips, and accessible drug treatment for combined opioid and stimulant use, are urgently needed in the rural U.S. to reduce overdose and other adverse outcomes.

Barriers to opioid use disorder treatment among people who use drugs in the rural United States: A qualitative, multi-site study.

BACKGROUND: In 2020, 2.8 million people required substance use disorder (SUD) treatment in nonmetropolitan or 'rural' areas in the U.S. Among this population, only 10% received SUD treatment from a specialty facility, and 1 in 500 received medication for opioid use disorder (MOUD). We explored the context surrounding barriers to SUD treatment in the rural United States. METHODS: We conducted semi-structured, in-depth interviews from 2018 to 2019 to assess barriers to SUD treatment among people who use drugs (PWUD) across seven rural U.S. study sites. Using the social-ecological model (SEM), we examined individual, interpersonal, organizational, community, and policy factors contributing to perceived barriers to SUD treatment. We employed deductive and inductive coding and analytical approaches to identify themes. We also calculated descriptive statistics for participant characteristics and salient themes. RESULTS: Among 304 participants (55% male, mean age 36 years), we identified barriers to SUD treatment in rural areas across SEM levels. At the individual/interpersonal level, relevant themes included: fear of withdrawal, the need to "get things in order" before entering treatment, close-knit communities and limited confidentiality, networks and settings that perpetuated drug use, and stigma. Organizational-level barriers included: strict facility rules, treatment programs managed like corrections facilities, lack of gender-specific treatment programs, and concerns about jeopardizing employment. Community-level barriers included: limited availability of treatment in local rural communities, long distances and limited transportation, waitlists, and a lack of information about treatment options. Policy-level themes included insurance challenges and system-imposed barriers such as arrest and incarceration. CONCLUSION: Our findings highlight multi-level barriers to SUD treatment in rural U.S. communities. Salient barriers included the need to travel long distances to treatment, challenges to confidentiality due to small, close-knit communities where people are highly familiar with one another, and high-threshold treatment program practices. Our findings point to the need to facilitate the elimination of treatment barriers at each level of the SEM in rural America.

Hereditary Haemorrhagic Telangiectasia (HHT) Marked by ACVRL1C1120T Variant Displays Hypopigmented Naevi and Frequent Bleeding Episodes if CYP2C9 Co-Mutated: Clinical Notes & Rationale of Patient Registry.

Hereditary haemorrhagic telangiectasia (HHT) exhibits considerable phenotypic heterogeneity. Therefore, precise mutation screening and evaluation of patient risk must be determined in every HHT family. We present an HHT-2 case with an initial life-threatening bleeding episode that led to identification of a relatively large HHT family. Exome sequencing of the family members determined HHT-associated ACVRL1C1120T variant resulting in Arg374Trp substitution at the Ser/Thr-kinase domain region. The affected members display typical epistaxis symptomatology from early childhood resulting in sideropoenia. In addition, the HHT patients also displayed dermatology findings such as facial teleangiectasias and trunk/limb white spots representing post-inflammatory hypopigmentation. Interestingly, co-segregating with modifying cytochrome P450 (CYP2C) variant in the HHT patients led to NSAID intolerance marked by increased frequency of bleeding episodes. No arterial-venous malformation of the visceral organs and brain or association with cancer were observed. The heterogeneity of clinical presentation and the role of other variants support the need of regular patient monitoring and development of a nation-wide patient registry.

A genotypic and spatial epidemiologic analysis of Massachusetts' Mycobacterium tuberculosis cases from 2012 to 2015.

BACKGROUND: Massachusetts had a rate of 2.8 cases of tuberculosis (TB) per 100,000 individuals in 2015. Although TB in Massachusetts is on the decline, the case rate remains far above the 2020 National TB Target of 1.4 per 100,000. To reduce the TB case rate in Massachusetts, it is necessary to understand the local epidemiology and transmission risks. METHODS: We used an existing TB case database of Massachusetts TB cases in the time frame from 2012 to 2015, which links de-identified patient demographic information with TB genotypes obtained from the United States Centers for Disease Control and Prevention's (CDC) TB Genotyping Information Management System database. Two or more cases with identical genotypes, which were close in space (within 50 km), as determined in a geographic information system (GIS), and time (3 years), were considered TB clusters. RESULTS: We analyzed 543 genotyped cases. We identified a total of 85 cases that met the TB cluster criteria, and a total of 33 clusters. US-born individuals (p = 0.003), homeless individuals (p = 0.001) and those reporting illicit substance use (p = 0.001) and alcohol use (p = 0.001) were more likely to appear in a TB cluster. CONCLUSION: Through a combined genotypic and spatial epidemiological approach, we identified populations and individuals more likely to be in a TB cluster. Testing populations identified as at risk for being in a TB cluster, and providing appropriate treatment, may decrease the overall TB case rate and support efforts to achieve national 2020 TB targets.

Modeling cancer driver events in vitro using barrier bypass-clonal expansion assays and massively parallel sequencing.

The information on candidate cancer driver alterations available from public databases is often descriptive and of limited mechanistic insight, which poses difficulties for reliable distinction between true driver and passenger events. To address this challenge, we performed in-depth analysis of whole-exome sequencing data from cell lines generated by a barrier bypass-clonal expansion (BBCE) protocol. The employed strategy is based on carcinogen-driven immortalization of primary mouse embryonic fibroblasts and recapitulates early steps of cell transformation. Among the mutated genes were almost 200 COSMIC Cancer Gene Census genes, many of which were recurrently affected in the set of 25 immortalized cell lines. The alterations affected pathways regulating DNA damage response and repair, transcription and chromatin structure, cell cycle and cell death, as well as developmental pathways. The functional impact of the mutations was strongly supported by the manifestation of several known cancer hotspot mutations among the identified alterations. We identified a new set of genes encoding subunits of the BAF chromatin remodeling complex that exhibited Ras-mediated dependence on PRC2 histone methyltransferase activity, a finding that is similar to what has been observed for other BAF subunits in cancer cells. Among the affected BAF complex subunits, we determined Smarcd2 and Smarcc1 as putative driver candidates not yet fully identified by large-scale cancer genome sequencing projects. In addition, Ep400 displayed characteristics of a driver gene in that it showed a mutually exclusive mutation pattern when compared with mutations in the Trrap subunit of the TIP60 complex, both in the cell line panel and in a human tumor data set. We propose that the information generated by deep sequencing of the BBCE cell lines coupled with phenotypic analysis of the mutant cells can yield mechanistic insights into driver events relevant to human cancer development.

Metal free carboamination of internal alkynes--an easy access to polysubstituted quinolines.

A metal free carboamination of unactivated alkynes towards highly substituted quinolines was realized in the presence of a synergistic Brønsted acid catalyst system. Supported by mechanistic probes, the reaction proceeds via a highly reactive vinyl cation in a C-C bond formation--Schmidt reaction sequence. The irreversible extrusion of N2, as a powerful driving force, allows for a general conversion of poorly nucleophilic aliphatic alkynes.

Aggressive acute myeloid leukemia in PU.1/p53 double-mutant mice.

PU.1 downregulation within hematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to their aggressiveness; however, its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1(ure/ure) mice (PU.1(ure/ure)p53(-/-)) results in more aggressive AML with shortened overall survival. PU.1(ure/ure)p53(-/-) progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1 levels in PU.1(ure/ure)p53(-/-) mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved in the pathogenesis of AML and its aggressiveness characterized by p53 mutation.

5-azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity.

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte-macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis.

Hematopoiesis is coordinated by a complex regulatory network of transcription factors and among them PU.1 (Spi1, Sfpi1) represents a key molecule. This review summarizes the indispensable requirement of PU.1 during hematopoietic cell fate decisions and how the function of PU.1 can be modulated by protein-protein interactions with additional factors. The mutual negative regulation between PU.1 and GATA-1 is detailed within the context of normal and leukemogenic hematopoiesis and the concept of 'differentiation therapy' to restore normal cellular differentiation of leukemic cells is discussed.

TRAIL (Apo2L) suppresses growth of primary human leukemia and myelodysplasia progenitors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, APO2L) has been shown to induce apoptosis in a number of tumor cell lines as well as in some primary tumors whereas cells from most normal tissues are highly resistant to TRAIL-induced apoptosis. We have studied the susceptibility of primary malignant and normal bone marrow hematopoietic progenitors to TRAIL-induced apoptosis. Extracellular domain of human TRAIL with N-terminal His(6) tag (His-TRAIL, amino acids 95-281) was produced in E. coli and its apoptosis-inducing ability was compared with the leucine-zipper containing TRAIL, LZ-TRAIL. Both variants of TRAIL had the same apoptosis-inducing ability. Clonogenic progenitor assays showed that His-TRAIL significantly reduced the number of myeloid colonies (CFU-GM) and clusters from patients with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS). His-TRAIL had no negative effect on the number of CFU-GM colonies and clusters derived from bone marrow cells of AML patients in complete remission, and lymphoma patients without bone marrow involvement, as well as those derived from normal cord blood cells. Moreover, we found that normal human stem cells treated with high doses of His-TRAIL maintain a repopulating potential when transplanted into NOD/SCID mice. To conclude, our data document that TRAIL does not affect normal human hematopoiesis but suppresses the growth of early primary leukemia and myelodysplasia progenitors.

Angiopoietin-1, angiopoietin-2 and Tie-2 in tumour and non-tumour tissues during growth of experimental melanoma.

Tumour progression is dependent on the formation of new vessels in tumour tissue. Tumour cells produce a variety of factors that influence vessel growth and maintenance both in tumour and tumour-adjacent tissues. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their tyrosine kinase receptor Tie-2 have been shown to play an important role in the processes of growth and remodelling of normal as well as tumour vessels. We studied gene expression of the angiogenic factors Ang-1 and Ang-2 and of their tyrosine kinase receptor Tie-2 in the tumour and non-tumour tissues of mice bearing the experimental melanoma B16. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we measured Ang-1, Ang-2 and Tie-2 mRNA levels in the tumour, bone marrow, liver and spleen. Melanoma tissue overexpressed Ang-2 mRNA compared with spleen, liver and bone marrow of normal mice, suggesting its role during melanoma progression. On the other hand, there was a significant decrease in Ang-2 mRNA level in bone marrow cells collected on days 5 and 10 of tumour growth compared with the expression of Ang-2 mRNA in the bone marrow of normal mice and those collected on days 15 and 20 of tumour growth. These data demonstrate, for the first time, an ectopic effect of the tumour on the gene coding for an angiogenic factor, and also suggest that tumour growth may influence angiogenesis and/or vasculogenesis in distant organs.

Diagnosis of polycythemia vera in an anemic patient.

Criteria proposed by the Polycythemia Vera Study Group (PVSG) as well as several derived algorithms are currently used for the diagnosis of polycythemia vera. Although these guidelines have significantly enhanced diagnostic accuracy, they uniformly consider erythrocytosis as the requisite premise for instigating the subsequent workup. We describe the unusual presentation of a patient with microcytic anemia in whom the diagnosis of polycythemia vera was reached using the PVSG criteria and confirmed by in vitro culture assay of erythroid progenitor cells. This case highlights the usefulness of the PVSG criteria, including the red cell mass determination, for the diagnosis of polycythemia vera even in anemic patients. The roles of spleen red cell pooling and plasma volume expansion as major determinants of this unusual presentation are discussed.

The social geography of AIDS and hepatitis risk: qualitative approaches for assessing local differences in sterile-syringe access among injection drug users.

While significant gains have been achieved in understanding and reducing AIDS and hepatitis risks among injection drug users (IDUs), it is necessary to move beyond individual-level characteristics to gain a fuller understanding of the impact of social context on risk. In this study, 6 qualitative methods were used in combination with more traditional epidemiologic survey approaches and laboratory bioassay procedures to examine neighborhood differences in access to sterile syringes among IDUs in 3 northeastern cities. These methods consisted of (1) neighborhood-based IDU focus groups to construct social maps of local equipment acquisition and drug use sites; (2) ethnographic descriptions of target neighborhoods; (3) IDU diary keeping on drug use and injection equipment acquisition; (4) ethnographic day visits with IDUs in natural settings; (5) interviews with IDUs about syringe acquisition and collection of syringes for laboratory analysis; and (6) focused field observation and processual interviewing during drug injection. Preliminary findings from each of these methods are reported to illustrate the methods' value in elucidating the impact of local and regional social factors on sterile syringe access.

Chromatin remodeling gene SMARCA5 is dysregulated in primitive hematopoietic cells of acute leukemia.

We identified a subset of genes involved in chromatin remodeling whose mRNA expression changes in differentiating mouse erythroleukemia (MEL) cells. We furthermore tested their mRNA expression patterns in normal and malignant CD34+ bone marrow cells. SMARCA5, imitation switch gene homologue, was rapidly silenced during in vitro erythroid differentiation of MEL cells whereas it was up-regulated in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Moreover, SMARCA5 mRNA levels decreased in AML CD34+ progenitors after the patients achieved complete hematologic remission. We detected high levels of SMARCA5 mRNA in murine bone marrow and spleen and monitored its expression in these hematopoietic tissues during accelerated hematopoiesis following hemolytic anemia induced by phenylhydrazine. SMARCA5 expression levels decreased after the onset of accelerated erythropoiesis. Our data suggest that both in vitro and in vivo induction of differentiation is followed by down-regulation of SMARCA5 expression. In CD34+ AML progenitors over-expression of SMARCA5 may thus dysregulate the genetic program required for normal differentiation.

Ribosomal protein S19 gene mutations in patients with diamond-blackfan anemia and identification of ribosomal protein S19 pseudogenes.

Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell hypoplasia characterized by a selective defect of erythropoiesis with a normochromic macrocytic anemia and reticulocytopenia often accompanied by various congenital anomalies. The critical region responsible for the pathogenesis of DBA has been mapped in some patients to chromosome 19q13.2 (P Gustavsson, E Garelli, N Draptchinskaia, et al. Am. J. Hum. Genet. 63:1388-1395, 1998) and the gene encoding ribosomal protein S19 (RPS19) is believed to be the candidate gene. Here we present molecular analysis of the RPS19 gene in DBA patients from the Czech National DBA Registry. We found that the RPS19 gene was mutated in 25% (5/20) of DBA patients (insertion, deletion, and point mutations, but no nonsense or splice site mutations). Point mutations were localized to hot spots defined by Willig (TN Willig, N Draptchinskaia, I Dianzani, et al. Blood 94:4294-4306, 1999). Moreover, we describe two processed RPS19 pseudogenes, which were not expressed. Possible models of the DBA pathogenesis in the view of RPS19 mutations are discussed.

Guinea pig serum erythropoietin (EPO) selectively stimulates guinea pig erythroid progenitors: human or mouse erythroid progenitors do not form erythroid burst-forming unit colonies in response to guinea pig serum EPO.

Erythropoietin (EPO) is the primary regulator of mammalian erythropoiesis, providing a proliferative and differentiative signal to the early EPO-responsive erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid, as well as to later EPO-responsive erythroid progenitors. EPO is secreted by the kidney in response to hypoxia and anemia. There is an extensive biological crossreactivity between human EPO and the EPOs of other mammals. Necas et al. have reported that this crossreactivity may not include the guinea pig (Cavia porcelllus). Because the specificity of the guinea pig's erythropoietic responses may be of biological significance, we compared guinea pig hypoxic serum with mouse (m) and human (h) recombinant (r) EPOs for their ability to induce erythroid progenitor proliferation and differentiation in semisolid cultures. Guinea pig bone marrow mononuclear cells (BMMCs) formed BFU-E colonies in response to guinea pig hypoxic serum, rhEPO, or rmEPO in a dose-dependent fashion. Neither human nor mouse BMMCs responded to guinea pig hypoxic serum; however, guinea pig hypoxic serum exerted no inhibitory effect on human or mouse in vitro erythroid differentiation in the presence of rhEPO or rmEPO. The intensity of the EPO band on Western blotting analysis of guinea pig hypoxic serum was significantly greater than in nonhypoxic serum. This suggests that guinea pig erythropoiesis is mediated by EPO and stimulated by hypoxia in a fashion similar to that observed in human and mouse erythropoiesis. Furthermore, guinea pig EPO did not stimulate human or mouse erythroid differentiation in vitro, whereas guinea pig erythroid progenitors could be stimulated by human or mouse EPO, suggesting structural differences in guinea pig EPO and EPO receptor (EPOR) compared with human or mouse EPO and EPOR. These differences probably evolved after the guinea pig's ancestors diverged from myomorph rodents. Further characterization of the guinea pig EPO and EPOR should facilitate our understanding of the interaction between EPO and EPOR.

Losartan, an angiotensin II type 1 receptor antagonist, lowers hematocrit in posttransplant erythrocytosis.

The mechanism by which angiotensin-converting enzyme inhibitors reduce red cell mass in renal transplant recipients with erythrocytosis is unclear. To examine the role of angiotensin II in this disorder, losartan (a competitive antagonist of the angiotensin II type 1 [AT1] receptor) was administered to 23 patients with erythrocytosis. Fourteen patients took 25 mg/d for 8 wk; nine others were treated with 50 mg/d for 8 wk. Hematocrit decreased from 0.527 +/- 0.027 to 0.487 +/- 0.045 after 8 wk (P < 0.01)--by at least 0.04 in 19 patients. Decrement in hematocrit in the initial 8 wk of therapy was significantly greater in patients administered 50 mg/d than in patients on 25 mg/d. Twelve of 14 patients initially treated with 25 mg/d showed a small change in hematocrit; the dose was increased to 50 mg/d for 8 more wk. Hematocrit decreased from 0.528 +/- 0.030 before losartan treatment to 0.483 +/- 0.055 after 16 wk (P < 0.01). After therapy, serum erythropoietin significantly decreased in eight patients with elevated baseline levels, but not in 15 patients with normal baseline levels; however, hematocrit significantly decreased in both groups. Losartan was withdrawn in 16 patients; hematocrit increased from 0.440 +/- 0.057 to 0.495 +/- 0.049 after 8.9 +/- 7.5 wk (P < 0.001), without change in serum erythropoietin. Thus, specific blockade of AT1 receptors inhibited erythropoiesis, suggesting a pathogenic role for angiotensin II in posttransplant erythrocytosis.