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In 2020, the Office of Minority Health (OMH) funded the establishment of the National COVID-19 Resiliency Network (NCRN) to engage priority communities that included African American, American Indian, Alaska Native, Asian, Hispanic, Native Hawaiian, and Pacific Islander persons to aggregate culturally relevant messages, education tools, and national data sources. A mobile application and Web site supported a culturally sensitive, equity-focused communication and dissemination initiative to link communities to COVID-19 clinical, social, and behavioral health services to address health determinants. ( Am J Public Health . 2024;114(S9): S718-S722. https://doi.org/10.2105/AJPH.2024.307813).

Objective: This study assesses the collective community capacity of the National COVID-19 Resiliency Network (NCRN), a multisectoral network mitigating the disproportionate impact of COVID-19 on minoritized populations., Methods: From January to April 2022, we used two concurrent data collection methods: a Collective Community Capacity (C3) survey (n=65) and key informant interviews (KIIs) (n=26). The C3 assessed capacity for creation of a shared vision, engagement in community change, and distributive leadership. KIIs assessed perspectives on network formation and implementation. We used a convergent design and triangulation for interpretation., Results: NCRN has growing collective community capacity. The C3 survey found high capacity for establishing a shared mission and evidence of mutual commitment, trust, and accountability. About three-quarters of respondents strongly agreed that partners addressed social, economic, and cultural barriers related to COVID-19. Interviewees valued NCRN leaders' openness, availability, and willingness to listen. Partners learned from one another, increased their health communication capacity, and supported sustainability. They sought greater opportunities to partner and support decision-making., Conclusions: NCRN developed a collaborative network with a shared vision of improving health equity during and beyond the COVID-19 pandemic, while identifying areas for improvement in distributive leadership. Findings can support other organizations seeking to build collective community capacity to address equity in public health emergencies.

Objective: To examine the association between county-level Black-White residential segregation and COVID-19 vaccination rates., Design: Observational cross-sectional study using multivariable generalized linear models with state fixed effects to estimate the average marginal effects of segregation on vaccination rates., Setting: National analysis of county-level vaccination rates., Main Outcome Measure: County-level vaccination rates across the United States., Results: We found an overall positive association between county-level segregation and the proportion population fully vaccinated, with a 6.8, 11.3, and 12.8 percentage point increase in the proportion fully vaccinated by May 3, September 27, and December 6, 2021, respectively. Effects were muted after adjustment for sociodemographic variables. Furthermore, in analyses including an interaction term between the county proportion of Black residents and the county dissimilarity index, the association between segregation and vaccination is positive in counties with a lower proportion of Black residents (ie, 5%) but negative in counties with the highest proportions of Black residents (ie, 70%)., Conclusions: Findings highlight the importance of methodological decisions when modeling disparities in COVID-19 vaccinations. Researchers should consider mediating and moderating factors and examine interaction effects and stratified analyses taking racial group distributions into account. Results can inform policies around the prioritization of vaccine distribution and outreach., Competing Interests: The authors have no conflicts of interest to report., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc.)

Background: Interpersonal primary care continuity or chronic condition continuity (CCC) is associated with improved health outcomes. Ambulatory care-sensitive conditions (ACSC) are best managed in a primary care setting, and chronic ACSC (CACSC) require management over time. However, current measures do not measure continuity for specific conditions or the impact of continuity for chronic conditions on health outcomes. The purpose of this study was to design a novel measure of CCC for CACSC in primary care and determine its association with health care utilization., Methods: We conducted a cross-sectional analysis of continuously enrolled, nondual eligible adult Medicaid enrollees with a diagnosis of a CACSC using 2009 Medicaid Analytic eXtract files from 26 states. We conducted adjusted and unadjusted logistic regression models of the relationship between patient continuity status and emergency department (ED) visits and hospitalizations. Models were adjusted for age, sex, race/ethnicity, comorbidity, and rurality. We defined CCC for CACSC as at least 2 outpatient visits with any primary care physician for a CACSC in the year, and (2) more than 50% of outpatient CACSC visits with a single PCP., Results: There were 2,674,587 enrollees with CACSC and 36.3% had CCC for CACSC visits. In fully adjusted models, enrollees with CCC were 28% less likely to have ED visits compared with those without CCC (aOR = 0.71, 95% CI = 0.71 - 0.72) and were 67% less likely to have hospitalization than those without CCC (aOR = 0.33, 95% CI = 0.32-0.33)., Conclusions: CCC for CACSCs was associated with fewer ED visits and hospitalizations in a nationally representative sample of Medicaid enrollees., Competing Interests: Conflicts of interest: None., (© Copyright by the American Board of Family Medicine.)

Introduction: Over the last 30 years, the adoption of health information technology and digital health tools (DHTs) into the US health system has been instrumental to improving access to care, especially for people living in rural, underserved, and underrepresented communities. Despite widespread adoption of DHTs by primary care clinicians, documented challenges have contributed to inequitable use and benefit. The COVID-19 pandemic necessitated rapid adoption of DHTs, accelerated by state and federal policy changes, in order to meet patient needs and ensure access to care., Methods: The Digital Health Tools Study employed a mixed methods approach to assess adoption and use of DHTs by primary care clinicians in southeastern states and identify individual- and practice-level barriers and facilitators to DHT implementation. A survey was conducted using a multi-modal recruitment strategy: newsletters, meeting/conference presentations, social media, and emails/calls. Focus groups were conducted to assess priorities, barriers, and facilitators and were recorded/transcribed verbatim. Descriptive statistics were calculated for survey results, produced for the whole sample, and stratified by state. Thematic analysis was conducted of focus group transcripts., Results: There were 1215 survey respondents. About 55 participants who had missing demographic information were excluded from the analysis. About 99% of clinicians used DHTs in the last 5 years, modalities included: telehealth (66%), electronic health records (EHRs; 66%), patient portals (49%), health information exchange (HIE; 41%), prescription drug monitoring programs (39%), remote/home monitoring (27%), and wearable devices (22%). Time (53%) and cost (51%) were identified as barriers. About 61% and 75% of clinicians reported being "satisfied" to "very satisfied" with telemedicine and EHRs, respectively. Seven focus groups with 25 clinicians were conducted and indicated COVID-19 and the use of supplemental tools/apps to connect patients to resources as major motivators for adopting DHTs. Challenges included incomplete and difficult-to-utilize HIE interfaces for providers and internet/broadband access and poor connectivity for patients., Conclusions: This study describes the impact adopting DHTs by primary care clinicians has on expanded access to healthcare and reducing health disparities in regions with longstanding health and social inequities. The findings identify opportunities to leverage DHTs to advance health equity and highlight opportunities for policy improvement.

Context: There is a need to understand population race and ethnicity disparities in the context of sociodemographic risk factors in the US experience of the COVID-19 pandemic., Objective: Determine the association between county-level proportion of non-Hispanic Black (NHB) on county COVID-19 case and death rates and observe how this association was influenced by county sociodemographic and health care infrastructure characteristics., Design and Setting: This was an ecologic analysis of US counties as of September 20, 2020, that employed stepwise construction of linear and negative binomial regression models. The primary independent variable was the proportion of NHB population in the county. Covariates included county demographic composition, proportion uninsured, proportion living in crowded households, proportion living in poverty, population density, state testing rate, Primary Care Health Professional Shortage Area status, and hospital beds per 1000 population., Main Outcome Measures: Outcomes were exponentiated COVID-19 cases per 100 000 population and COVID-19 deaths per 100 000 population. We produced county-level maps of the measures of interest., Results: In total, 3044 of 3142 US counties were included. Bivariate relationships between the proportion of NHB in a county and county COVID-19 case (Exp β = 1.026; 95% confidence interval [CI], 1.024-1.028; P < .001) and death rates (rate ratio [RR] = 1.032; 95% CI, 1.029-1.035; P < .001) were not attenuated in fully adjusted models. The adjusted association between the proportion of NHB population in a county and county COVID-19 case was Exp β = 1.025 (95% CI, 1.023-1.027; P < .001) and the association with county death rates was RR = 1.034 (95% CI, 1.031-1.038; P < .001)., Conclusions: The proportion of NHB people in a county was positively associated with county COVID-19 case and death rates and did not change in models that accounted for other socioecologic and health care infrastructure characteristics that have been hypothesized to account for the disproportionate impact of COVID-19 on racial and ethnic minority populations. Results can inform efforts to mitigate the impact of structural racism of COVID-19., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Rulemaking is one of the most important ways the federal government makes public policy. It frequently has significant impact on individuals, communities, and organizations. Yet, few of those directly affected are familiar with the rulemaking process, and even fewer understand how it works. This article describes a case study of the Transdisciplinary Collaborative Center for Health Disparities Research Health Information Technology (TCC HIT) Policy Project's approach to health-policy engagement using: 1) social media; and 2) a webinar to educate stakeholders on the rulemaking process and increase their level of meaningful engagement with the Medicare Access and CHIP Reauthorization Act of 2015 (MACRA) proposed rule public comment submission. The webinar "Paying for Quality: What Is the Impact on Health Equity" was promoted through Twitter and held in June 2016. In total, we posted 19 tweets using two distinct hashtags (#MACRA4Equity, #MACRA2Equity) to raise awareness of the upcoming MACRA proposed rule and its possible effects on health equity. Overall, 252 individuals registered for the webinar, and more than half participated (n=133). Most (67%) registrants reported that health policy was not the primary focus of their current position. Based on information provided in the webinar, 95% agreed that their understanding of the topic improved. By the end of the webinar, 44% of participants indicated that they planned to submit public comments for MACRA, a 12% increase compared with those who planned to submit at the time of registration. The TCC health-policy engagement strategy demonstrates the feasibility of engaging a diverse audience around health policy issues, particularly those who are not typically engaged in policy work., Competing Interests: Competing Interests: None declared.

Objective: This study characterized telemedicine utilization among Medicaid enrollees by patients' demographic characteristics, geographic location, enrollment type, eligibility category, and clinical conditions., Methods: This study used 2008-2009 Medicaid claims data from 28 states and the District of Columbia to characterize telemedicine claims (indicated by GT for professional fee claims or Q3014 for facility fees) on the basis of patients' demographic characteristics, geographic location, enrollment type, eligibility category, and clinical condition as indicated by ICD-9 codes. States lacking Medicaid telemedicine reimbursement policies were excluded. Chi-square tests were used to compare telemedicine utilization rates and one-way analysis of variance was used to estimate mean differences in number of telemedicine encounters among subgroups., Results: A total of 45,233,602 Medicaid enrollees from the 22 states with telemedicine reimbursement policies were included in the study, and .1% were telemedicine users. Individuals ages 45 to 64 (16.4%), whites (11.3%), males (8.5%), rural residents (26.0%), those with managed care plans (7.9%), and those categorized as aged, blind, and disabled (28.1%) were more likely to receive telemedicine (p<.001). Nearly 95% of telemedicine claims were associated with a behavioral health diagnosis, of which over 50% were for bipolar disorder and attention-deficit disorder or attention-deficit hyperactivity disorder (29.3% and 23.4%, respectively). State-level variation was high, ranging from .0 to 59.91 claims per 10,000 enrollees (Arkansas and Arizona, respectively)., Conclusions: Despite the touted potential for telemedicine to improve health care access, actual utilization of telemedicine in Medicaid programs was low. It was predominantly used to treat behavioral health diagnoses. Reimbursement alone is insufficient to support broad utilization for Medicaid enrollees.

Background: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome., Methods: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon., Results: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate., Conclusion: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

We have utilized oligonucleotide microarrays to identify novel genes of potential clinical and biological importance in prostate cancer. RNA from 74 prostate cancers and 164 normal body samples representing 40 different tissues were analysed using a customized Affymetrix GeneChip oligonucleotide microarray representative of over 90% of the expressed human genome. The gene for the zinc transporter ZnT4 was one of several genes that displayed significantly higher expression in prostate cancer compared to normal tissues from other organs. A polyclonal antipeptide antibody was used to demonstrate ZnT4 expression in the epithelium of all 165 elements of benign and 326 elements of localized prostate cancers examined and in nine of 10 advanced prostate cancer specimens by immunohistochemistry. Interestingly, decreased intensity of ZnT4 immunoreactivity occurred in the progression from benign to invasive localized prostate cancer and to metastatic disease. Immunofluorescence analysis and surface biotinylation studies of cells expressing ZnT4 localised the protein to intracellular vesicles and to the plasma membrane. These findings are consistent with a role for ZnT4 in vesicular transport of zinc to the cell membrane and potentially in efflux of zinc in the prostate.

Current models of prostate cancer classification are poor at distinguishing between tumors that have similar histopathological features but vary in clinical course and outcome. Here, we applied classical survival analysis to genome-wide gene expression profiles of prostate cancers and preoperative prostate-specific antigen (PSA) levels from each patient, to identify prognostic markers of disease relapse that provide additional predictive value relative to PSA concentration. Three of approximately 200 probesets showing strongest correlation with relapse were identified as the gene for the putative calcium channel protein, trp-p8, with loss of trp-p8 mRNA expression associated with a significantly shorter time to PSA relapse-free survival. We observed subsequently that trp-p8 is lost in the transition to androgen independence in a prostate cancer xenograft model and in prostate cancer tissue from patients treated preoperatively with antiandrogen therapy, suggesting that trp-p8 is androgen regulated, and its loss may be associated with more advanced disease. The identification of trp-p8 and other proteins implicated in the phosphatidylinositol signal transduction pathway that are associated with prostate cancer outcome, both here and in other published work, suggests an integral role for this pathway in prostate carcinogenesis. Thus, our findings demonstrate that multivariable survival analysis can be applied to gene expression profiles of prostate cancers with censored follow-up data and used to identify molecular markers of prostate cancer relapse with strong predictive power and relevance to the etiology of this disease.

gammadelta T lymphocytes in the intestinal intraepithelial layer (gammadelta IELs) are thought to contribute to immune competence, but their actual function remains poorly understood. Here we used DNA microarrays to study the gene expression profile of gammadelta IELs in a Yersinia infection system to better define their roles. To validate this approach, mesenteric lymph node CD8(+) alphabeta T cells were similarly analyzed. The transcription profiles show that, whereas lymph node CD8(+) alphabeta T cells must be activated to become cytotoxic effectors, gammadelta IELs are constitutively activated and appear to use different signaling cascades. Our data suggest that gammadelta IELs may respond efficiently to a broad range of pathological situations irrespective of their diverse T cell antigen receptor repertoire. gammadelta IELs may modulate local immune responses and participate in intestinal lipid metabolism, cholesterol homeostasis, and physiology. This study provides a strong basis for further investigations of the roles of these cells as well as mucosal immune defense in general.

Self-tolerance is achieved by deleting or regulating self-reactive lymphocytes at a series of cellular checkpoints placed at many points along the developmental pathways to plasma cells and effector T cells. At each checkpoint, what are the molecular pathways that determine whether a lymphocyte remains quiescent, begins dividing, differentiates or dies? In splenic B cells, the decision between quiescence, tolerance by anergy, and activation provides a tractable setting to explore these issues by global gene expression profiling on DNA microarrays. Here we discuss the application of microarrays to illuminate a set of cell fate decisions that appear to be determined by summation of numerous small changes in expression of stimulatory and inhibitory genes. Many genes with known or predicted inhibitory functions are highly expressed in naive, quiescent B cells, notably the signal inhibitor SLAP and DNA-binding proteins of the Kruppel family (LKLF, BKLF, GKLF), Tsc-22, GILZ, Id-3, and GADD45. Activation of naive B cells, triggered by acute binding of antigen to the B-cell receptor, involves a rapid decrease in expression of these inhibitory genes. Promitotic genes are induced in parallel, including c myc, LSIRF/IRF4, cyclin D2, Egr-1 and Egr-2, as are the anti-apoptotic gene A1 and genes for the T-cell-attracting chemokines MIP-1alpha and beta. B-cell tolerance through the process of anergy, induced by chronic binding of self antigen, maintains expression of the inhibitory genes found in quiescent B cells and induces an additional set of inhibitory genes. The latter include inhibitors of signaling - CD72, neurogranin, pcp4 - and additional inhibitors of gene expression such as SATB1, MEF2C, TGIF and Nab-2. The effects of tolerance, the immunosuppressive drug FK506 and other modulators of calcium or MAPK signaling allow individual gene responses to be linked to different signal transduction pathways. The global molecular profiles obtained illustrate how quiescence and anergy are actively maintained in circulating B cells, how these states are switched to clonal expansion and how they could be better emulated by pro-tolerogenic drugs.

Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by gamma radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.

Therapy for transplant rejection, autoimmune disease and allergy must target mature lymphocytes that have escaped censoring during their development. FK506 and cyclosporin are immunosuppressants which block three antigen-receptor signalling pathways (NFAT, NFkappaB and JNK), through inhibition of calcineurin, and inhibit mature lymphocyte proliferation to antigen. Neither drug induces long-lived tolerance in vivo, however, necessitating chronic use with adverse side effects. Physiological mechanisms of peripheral tolerance to self-antigens provide an opportunity to emulate these processes pharmacologically. Here we use gene-expression arrays to provide a molecular explanation for the loss of mitogenic response in peripheral B-cell anergy, one aspect of immunological tolerance. Self-antigen induces a set of genes that includes negative regulators of signalling and transcription but not genes that promote proliferation. FK506 interferes with calcium-dependent components of the tolerance response and blocks an unexpectedly small fraction of the activation response. Many genes that were not previously connected to self-tolerance are revealed, and our findings provide a molecular fingerprint for the development of improved immunosuppressants that prevent lymphocyte activation without blocking peripheral tolerance.

p53 mutants in tumours have a reduced affinity for DNA and a reduced ability to induce apoptosis. We describe a mutant with the opposite phenotype, an increased affinity for some p53-binding sites and an increased ability to induce apoptosis. The apoptotic function requires transcription activation by p53. The mutant has an altered sequence specificity and selectively fails to activate MDM2 transcription. Loss of MDM2 feedback results in overexpression of the mutant, but the mutant kills better than wild-type p53 even in MDM2-null cells. Thus the apoptotic phenotype is due to a combination of decreased MDM2 feedback control and increased or unbalanced expression of other apoptosis-inducing p53 target genes. To identify these genes, DNA chips were screened using RNA from cells expressing the apoptosis-inducing mutant, 121F, and a sequence-specificity mutant with the reciprocal phenotype, 277R. Two potential new mediators of p53-dependent apoptosis were identified, Rad and PIR121, which are induced better by 121F than wild-type p53 and not induced by 277R. The 121F mutant kills untransformed MDM2-null but not wild-type mouse embryo fibroblasts and kills tumour cells irrespective of p53 status. It may thus expand the range of tumours which can be treated by p53 gene therapy.

We describe a novel gene, Blimp-1 (for B lymphocyte-induced maturation protein), transcripts of which are rapidly induced during the differentiation of B lymphocytes into immunoglobulin secretory cells and whose expression is characteristic of late B and plasma cell lines. The 856 amino acid open reading frame contains five Krüppel-type zinc finger motifs and proline-rich and acidic regions similar to those of known transcription factors. Serological studies show an approximately 100 kd protein that localizes to the nucleus. Stable or transient transfection of Blimp-1 into B cell lymphoma lines leads to the expression of many of the phenotypic changes associated with B cell differentiation into an early plasma cell stage, including induction of J chain message and immunoglobulin secretion, up-regulation of Syndecan-1, and increased cell size and granularity. Thus, Blimp-1 appears to be a pleiotropic regulatory factor capable of at least partially driving the terminal differentiation of B cells.

The p53 protein is apparently central to the development of human cancers because both alleles are often found to be mutated in different tumour types. In addition, wild-type p53 can inhibit transformation by viral and cellular oncogenes in vitro, so p53 has been classified as a tumour suppressor. Investigations of the normal function of p53 have indicated that at least one of its functions could involve the activation of gene expression through the binding of specific DNA-regulatory sequences. Also, overexpression of p53 can mediate growth arrest and repress transcription from a variety of promoters. We demonstrate here both in vivo and in vitro that expression of wild-type p53 specifically represses the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription is directed by a pyrimidine-rich initiator element, however, are immune to the effects of p53. Furthermore, we observe that repression is mediated by an interaction of p53 with basal transcription factor(s). Thus, p53 appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Human papillomavirus type 18 (HPV-18) infects genital squamous epithelium and is an etiological agent of cervical cancer. Cell-type-specific expression of HPV-18 is directed by the region upstream of the viral early genes that contains a transcriptional enhancer whose function is dependent solely on cellular factors. This element directs expression to high levels in squamous epithelial cells but is only weakly active in other cell types. We demonstrate by gel mobility-shift, methylation interference, and mutational analysis that the binding of two distinct factors to the enhancer is necessary for cell-type-specific transcriptional activation. One of these factors is identified as a keratinocyte-specific transcriptional activator, which we call KRF-1, while the other is a member of the AP-1 family. We also find that Oct-1 competes with KRF-1 for binding to enhancer sequences though it does not contribute to transcriptional activation. These results suggest a complex interplay of ubiquitous and cell-type-restricted transcriptional factors in the tissue- and differentiation-specific expression of HPV-18.

In vitro enzymatic amplification was applied to detect hepatitis B virus (HBV) DNA sequences in serum. This technique, known as the polymerase chain reaction (PCR) was used to amplify a 128 bp DNA fragment including a 112 nucleotide long sequence complementary to a region in the S gene of the HBV genome. Amplified samples were subjected to spot-test hybridization and scintillation counting using a 32P-labeled oligonucleotide probe. A kinetic study, performed for 4 to 32 PCR cycles with a viral particle preparation, showed a time-limited exponential accumulation of the specific amplified DNA fragment. Amplification yield after 32 cycles was at least 4 X 10(6) with a detection limit equal to 3 X 10(2) viral particles per ml of serum. As the reliability of the PCR technique was greatest for 24 PCR cycles, these conditions were used to develop a quantitative test with a detection limit of 4 X 10(4) viral particles per ml of serum. Results of this test were perfectly correlated with those obtained from the classical spot test without amplification. Ethidium bromide stained agarose gel and Southern blot analysis confirmed the specific amplification of the 128 bp HBV DNA fragment.

A segment of the largest open reading frame of hepatitis B virus (HBV) was inserted into an open reading frame vector directing the expression in Escherichia coli of a fusion molecule containing 143 HBV-encoded amino acids. The fusion protein was used to generate antiserum which served in immunoblots to identify a polypeptide with a molecular mass of 65 kilodaltons in HBV particles. Because of the small number of molecules in virus particles, unambiguous detection required the development of a highly sensitive immunoblot procedure.

Tests of small airway function (closing volume, forced expiratory flow in the middle half of the vital capacity, specific airway conductance, and peak expiratory flow) and routine spirometric tests (forced vital capacity, forced expiratory volume in one second, and residual volume) were performed in 848 adult black subjects of both sexes. The group included 422 smokers and 426 nonsmokers. No significant difference was found between smokers and nonsmokers for any of the tests, although some values were higher for men than for women. These findings suggest that reported abnormalities in small airway function in smokers in the general population may not be applicable to black smokers. This may be an important finding in ethnic variability in the incidence of chronic obstructive pulmonary disease.

Amino acid sequence similarity of the reverse transcriptases encoded by retroviruses and hepadnaviruses was first reported by Toh, H., Hayashida, H. & Miyata, T. (1983) Nature (London) 305, 827-829. The regions of similarity extend over a small number of amino acids and require the introduction of gaps through the open reading frame. By using an octapeptide region as the sole criterion for "taxonomic" classification, we have grouped the oncoviruses into two distinct categories and the lentiviruses and hepadnaviruses into two additional groupings. This classification suggests that murine and feline leukemia viruses may be more closely related to the viruses that are associated with leukemia in primates and cattle than had been appreciated. We have exploited a portion of this region because of the minimal translational codon degeneracy of the conserved residues. Unique oligonucleotides from this region have been designed and used in the primer-directed in vitro DNA amplification of the hepadnaviruses as a model system. In addition, mixtures of oligonucleotides with various sequences but of the same length were demonstrated to be efficient primers. The amplification procedure enabled dramatic increases in sensitivity and coincident detection of mammalian and avian genomes. This approach will be a valuable tool to detect and characterize members of viral groups. In addition, since short stretches of similarity have been frequently identified in related but distinct genes, such an approach could prove a valuable asset to molecular studies in general.

Human immunodeficiency virus (HIV) has been associated with acquired immunodeficiency syndrome and related disorders. Assays to detect antibodies to HIV proteins have been developed and used to screen sera for the identification of individuals who have been exposed to the virus. Although these serological tests have significant sensitivity and specificity for detecting exposure to the virus, they do not provide direct identification of HIV. We report here the application of recently developed nucleic acid amplification and oligonucleotide-based detection procedures for the identification of HIV sequences in established infected cell lines and in cells cultured from infected individuals.

By means of a selective DNA amplification technique called polymerase chain reaction, proviral sequences of the human immunodeficiency virus (HIV-1) were identified directly in DNA isolated from peripheral blood mononuclear cells (PBMCs) of persons seropositive but not in DNA isolated from PBMCs of persons seronegative for the virus. Primer pairs from multiple regions of the HIV-1 genome were used to achieve maximum sensitivity of provirus detection. HIV-1 sequences were detected in 100% of DNA specimens from seropositive, homosexual men from whom the virus was isolated by coculture, but in none of the DNA specimens from a control group of seronegative, virus culture-negative persons. However, HIV-1 sequences were detected in 64% of DNA specimens from seropositive, virus culture-negative homosexual men. This method of DNA amplification made it possible to obtain results within 3 days, whereas virus isolation takes up to 3 to 4 weeks. The method may therefore be used to complement or replace virus isolation as a routine means of determining HIV-1 infection.

A recombinant plasmid containing a 5.6-kilobase-pair DNA fragment of the Treponema pallidum genome was characterized by endonuclease mapping, and the encoded proteins were expressed in Escherichia coli and analyzed by use of in vitro transcription and translation. One of the proteins, identified as having a molecular weight of 37,000 (37K protein), was selected for further study. Initially, the seroreactivity of the partially purified 37K antigen was demonstrated by immunoblotting. After its purification to near homogeneity, the cloned T. pallidum protein was assessed for diagnostic significance by radioimmunoassay. Although first identified as seroreactive by screening with secondary syphilitic sera (T. E. Fehniger, A. M. Walfield, T. M. Cunningham, J. D. Radolf, J. N. Miller, and M. A. Lovett, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B156, p. 44), the antigen was shown to be serologically reactive with antibodies in serum from all stages of syphilis but was not recognized by serum from controls by both immunoblotting and radioimmune assay. Further, a monospecific polyclonal rabbit antiserum generated to the 37K antigen recognized a polypeptide of the same molecular weight from T. pallidum but did not efficiently recognize proteins from five nonpathogenic treponemes tested. Therefore, because of reactivity with and specificity for T. pallidum antibodies, the 37K antigen may be of serodiagnostic value in the detection of syphilis.