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Objective: This trial examined the effects of proximal/distal mediators and moderators of an Active Music Engagement (AME) intervention on young child/parent distress, quality of life, and family function outcomes. Methods: Child/parent dyads (n = 125) were randomized to AME or Audio-storybooks attention control condition. Each group received 3 sessions with a credentialed music therapist for 3 consecutive days with data collection at baseline, post-intervention (T2), and 30-days later (T3). Potential proximal mediators included within session child and parent engagement. Potential distal mediators included changes in perceived family normalcy, parent self-efficacy, and independent use of play materials. Potential moderators included parent/child distress with prior hospitalizations, parent traumatic stress screener (PCL-6), and child age. Outcomes included child emotional distress and quality of life; parent emotion, traumatic stress symptoms (IES-R), well-being; and family function. Mediation effects were estimated using ANCOVA, with indirect effects estimated using the percentile bootstrap approach. Moderation effects were tested by including appropriate interaction terms in models. Results: No significant mediation effects were observed. Child distress with prior hospitalizations moderated AME effects for IES-R intrusion subscale scores at T2 ( P = .01) and avoidance subscale scores at T3 ( P = .007). Traumatic stress screener scores (PCL-6) moderated intervention effects for IES-R hyperarousal subscale scores at T2 ( P = .01). There were no moderation effects for child age. Conclusions: AME is a promising intervention for mitigating traumatic stress symptoms and supporting well-being in parents of children with cancer, particularly for parents who screen high for traumatic stress and whose children are more highly distressed with hospitalization.

Periarticular needle-based therapies such as dry needling, acupuncture, and genicular nerve radiofrequency ablation are becoming more popular for treatment of knee osteoarthritis. These therapies are also being used after total knee arthroplasty for persistent postoperative pain. Although limited published evidence exists for the risk of periprosthetic joint infection after these procedures, we describe one case of periprosthetic joint infection developing shortly after dry needling and another case developing shortly after genicular nerve blocks. We present details of these 2 cases along with a review of the literature regarding the use of periarticular needle-based therapies after total knee arthroplasty.

Background: Race, gender, insurance status, and income play important roles in predicting health care outcomes. However, the impact of these factors has yet to be fully elucidated in the setting of hepatocellular carcinoma (HCC). Methods: We designed a retrospective cohort study utilizing data from the Surveillance, Epidemiology, and End Results (SEER) program to identify patients diagnosed with resectable HCC (N = 28,518). Demographic factors of interest included race (Asian/Pacific Islander [API], African American [AA], Native American/Alaska Native [NA], or White [WH]) and gender (male [M] or female [F]). Insurance classifications included those having Medicare/Private Insurance [ME/PI], Medicaid [MAID], or No Insurance [NI]. Median household income was estimated for all diagnosed with HCC. Endpoints included: (1) overall survival; (2) likelihood of receiving a recommendation for surgery; and (3) specific surgical intervention performed. Multivariate multinomial logistic regression for relative risk ratio (RRR) and Cox regression models were used to identify pertinent associations. Results: Race, gender, insurance status, and income had statistically significant effects on the likelihood of surgical recommendation and overall survival. API were more likely to receive a recommendation for hepatic resection (RRR = 1.45; 95% CI: 1.31-1.61; Reference Race: AA) and exhibited prolonged overall survival (HR = 0.77; 95% CI: 0.73-0.82; Reference Race: AA) as compared to members of any other ethnic group; there was no difference in these endpoints between AA, NA, or WH individuals. Gender also had a significant effect on survival: Females exhibited superior overall survival (HR = 0.89; 95% CI: 0.85-0.93; Reference Gender: M) as compared to males. Patients who had ME/PI were more likely than those with MAID or NI to receive a surgical recommendation. ME/PI was also associated with superior overall survival. Conclusions: Race, gender, insurance status, and income have measurable effects on HCC management and outcomes. The underlying causes of these disparities warrant further investigation.

Category: Trauma Introduction/Purpose: Traditional plate osteosynthesis for ankle fractures in a high risk patient population can predispose to complications as a result of the necessary soft tissue dissection and periosteal stripping. Complication rates among patients with multiple comorbidities has been shown to be as high as 30% in those who undergo traditional plate and screw fixation. 1 An alternative fixation construct, thought not widely used or recognized in the American literature is the locking intramedullary fibular nail. Intramedullary nails offer the theoretical advantages of stable fixation while utilizing smaller, percutaneous incisions and no periosteal stripping. The purpose of this study is to analyze the outcomes and complications of high risk patients who underwent ankle surgery using the intramedullary fibular nail. Methods: The medical records of all high risk patients who underwent ankle fracture fixation with a locking intramedullary fibular nail from 1/2011 through 6/2015 at our institution were reviewed. Fracture patterns included unstable lateral malleolar, bimalleolar, and trimalleolar fractures. Patients were considered high risk if they had medical comorbidities at the time of injury that have been shown in current literature to increase surgical complication risk. These comorbidities include neuropathy, uncontrolled diabetes, peripheral vascular disease, congestive heart failure, etc. We recorded patient demographics, fracture type, period of post-op NWB, time until discharge, time to weight bearing, complications, tourniquet time, size and length of nail implanted, and estimated blood loss. Attempts were made to contact all patients to complete a follow-up questionnaire regarding their satisfaction. Results: Twenty-two high risk patients with 23 ankle fracture were included. The average age was 64.8 years (SD 10.2, range 41- 82). The average follow up was 5.7 months (SD 5.7, range 2-30). There were no surgical site infections, wound complications, or non-unions. Two (8.7%) required a return to the operating room for symptomatic screw removal. The average tourniquet time was 35.8 minutes (SD 20.1, range 0-72) and the average operative time was 49.3 minutes (SD 14.3, range 25-70) respectively. The Olerud & Molander average score was 77.9 (SD 17.7, range 40-100). Fourteen of the twenty two patients could be contacted for a follow up questionnaire and 13 of the 14 (93%) said they were either very satisfied (64%) or satisfied (29%) with their injury outcome. Conclusion: The locking intramedullary fibular nail provides an alternative fixation technique that allows for protection of soft tissue, stable fracture fixation and satisfactory outcomes in the high risk patient population.

Cerebrospinal fluid (CSF) leak is the most commonly encountered perioperative complication in transsphenoidal surgery for pituitary lesions. Direct closure with a combination of autologous fat, local bone, and/or synthetic grafts remains the standard of care for leaks encountered at the time of surgery as well as postoperatively. The development of the vascularized nasoseptal flap as a closure technique has increased the surgeon’s capacity to correct even larger openings in the dura of the sella as well as widely exposed anterior skull base defects. Yet these advances in the technical nuances for management of post-transsphenoidal CSF leak are useless without the ability to recognize a CSF leak by physical examination, clinical history, biochemical testing, or radiographic assessment. Here, we report a case of a patient who developed a CSF leak 28 years after transsphenoidal surgery, precipitated by a robotic-assisted hysterectomy during which increased intra-abdominal pressure and steep Trendelenberg positioning were both factors. Given the remote nature of the patient’s transsphenoidal surgery and relative paucity of data regarding such a complication, the condition went unrecognized for several months. We review the available literature regarding risk and pathophysiology of CSF leak following abdominal surgery and propose the need for increased vigilance in identification of such occurrences with the increasing acceptance and popularity of minimally invasive abdominal and pelvic surgeries as standards in the field.

Background: Poor mental health among young adults in higher education is a growing concern. In recent years, the visibility of racism has sharply risen. Vicarious discrimination is defined as the secondhand witnessing of racism, and given society's increased accessibility to social media and the Internet, addressing indirect violence is urgently needed to inform anti-racism and mental health efforts. The current study examined associations between vicarious discrimination and mental health across a large sample of young college students in the United States., Methods: We analyzed data from the Healthy Minds Study (HMS; 2020-2021; N=130,566) and used multivariable logistic regression to examine whether past-year vicarious discrimination was associated with various mental health outcomes, adjusting for age, gender, race/ethnicity, and direct discrimination., Results: The sample was predominantly white (n=31,438, 63.66%) and female-identifying (n=34,313, 69.49%) with an average age of 21.1 years. Approximately 35.9% of the sample endorsed experiencing vicarious discrimination. Vicarious discrimination was associated with greater depression (OR:1.97; 95% CI: [1.86,2.09], p< 0.001), anxiety (OR:1.82; 95% CI: [1.72,1.92], p<0.001), languishing (OR:1.75; 95% CI: [1.65,1.87], p<0.001), perceived need for treatment (OR:2.24; 95% CI: [2.10,2.40], p<0.001), suicidal ideation (OR:1.86; 95% CI: [1.73,2.01], p<0.001), suicide plan (OR:1.91; 95% CI: [1.71,2.14], p<0.001), suicide attempt (OR:1.89; 95% CI: [1.51,2.36], p<0.001), self-injurious behavior (OR:2.0; 95% CI: [1.88,2.12], p<0.001), and loneliness (OR:1.67; 95% CI: [1.58,1.77], p<0.001)., Discussion: Consistent with growing literature, vicarious discrimination was associated with poorer mental health among young college students. Additional research should investigate moderators, mediators, and interventions to support those who may be impacted indirectly by discrimination., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest or competing interests that may inappropriately influence or bias this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The purpose of this study includes: 1) identifying classes of substance-related needs among Black women, and 2) examining the effect of substance-related need classes and culturally-relevant factors on Black women's use of substance use disorder (SUD) treatment and mutual support groups., Methods: As part of a longitudinal cohort study, Black women were recruited in prison nearing release, on probation, and in the community without involvement in the criminal legal system (CLS, n=565) and followed-up at 18-months. We conducted a baseline latent class analysis of substance-related needs among Black women. Logistic regression models adjusted for culturally-relevant factors to predict the use of treatment and frequency of mutual support group participation over 18-months among Black women who use drugs., Results: Four classes by level of needs were found: low, daily marijuana use, high mental health, and high comorbidity. During the 18-month follow-up, women characterized by the high comorbidity need class and with higher scores of religious well-being were more likely to frequently participate in mutual support groups. Non-CLS-involved women were less likely to engage with both treatment and mutual support groups than women from the prison sample at 18-months., Conclusions: This study highlights four distinct classes of substance-related needs among Black women, highlighting the complex patterns of behavior and within-racial group differences among Black women. Black women with high comorbidity needs were more likely to participate in mutual support groups, but the latent classes did not predict SUD treatment indicating other non-medical and social contextual need factors may be at play., Competing Interests: Declaration of Competing Interest No conflict declared., (Copyright © 2024. Published by Elsevier B.V.)

At the intersection of race and gender, Black men encounter conflicting and often stress-inducing gender norms. Research suggests Black men may utilize John Henryism Active Coping (JHAC), a culturally-relevant strategy to manage stress. However, little is known about how incarcerated Black men cope with gender role conflict (GRC) and resulting psychological distress. To better understand stressors and coping strategies among Black male prisoners, the current study examined the relationships between GRC, anxiety, and JHAC among N = 193 incarcerated Black men nearing community reentry. Hierarchical linear regression results showed length of incarceration, GRC, and anxiety were all negatively associated with JHAC. Full-time employment prior to incarceration was positively associated with JHAC. Moderation analyses indicated anxiety increased the strength of the negative association between GRC and JHAC. Implications highlight incarcerated Black men may benefit from interventions that encourage active coping strategies to manage gender-related stress and anxiety., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: Racial disparities in later-life depression among Americans are well-documented. Perceived discrimination has been linked to depressive symptoms among aging Black adults, but little research has considered how vicarious exposure to discrimination shapes the mental health of middle-aged and older Black adults., Methods: A subsample of Black adults aged 50-69 years ( N  = 273) were drawn from the Nashville Stress and Health Study. Lifetime vicarious exposures to major discrimination were assessed. Additionally, multivariate linear regression was employed to examine the association between vicarious experiences of major discrimination and depressive symptoms., Results: Once the Black adults in the sample had reached ages 50 and older, the most common discriminatory events that they had vicariously experienced in their lifetime occurred as a result of their loved ones' unfair interactions with law enforcement and the job market, respectively. Furthermore, our findings revealed that vicarious experiences of major discrimination were associated with higher levels of depressive symptoms among middle-aged and older Black adults., Conclusion: Vicarious, as well as personal, exposures to discrimination shape the mental health of Black Americans over the lifespan. The secondhand effects of discrimination must be considered for interventions aimed at reducing the mental health consequences of racism-related adversity as Black adults age.

During the pandemic, the overall mental health of the US population declined. Given higher rates of COVID-19 infections and deaths experienced by communities of color along with greater exposure to pandemic-related stressors (e.g., unemployment, food insecurity), we expect that the decline in mental health during the pandemic was more pronounced among Black, Hispanic, and Asian adults, with these groups also having less access to mental health services. We examine two nationally representative US surveys: the 2019 National Household Interview Survey (NHIS; N = 30,368) and the 2020-2021 Household Pulse Survey (HPS; N = 1,677,238). We find mental health of Black, Hispanic, and Asian respondents worsened relative to White respondents during the pandemic, with significant increases in depression and anxiety among racialized minorities compared to Whites. There is also evidence of especially high mental health burden for Black adults around the murder of George Floyd by police and for Asian adults around the murder of six Asian women in Atlanta. White respondents are most likely to receive professional mental health care before and during the pandemic, and Black, Hispanic, and Asian respondents demonstrate higher levels of unmet mental health care needs during the pandemic than White respondents. Our results indicate that within the current environment, White adults are at a large and systemic advantage buffering them from unexpected crises-like the COVID-19 pandemic. Without targeted interventions, the long-term social consequences of the pandemic and other co-occurring events (e.g., death of Black and Hispanic people by police) will likely include widening mental health disparities between racial/ethnic groups., (© 2022. W. Montague Cobb-NMA Health Institute.)

Background: The adverse mental health consequences of discrimination among Black adults, such as anxiety symptoms, are well documented. Prior research establishes anxiety as a risk factor for suboptimal health outcomes among Black adults. Most discrimination and mental health studies, however, have focused on the effects of personal experiences of discrimination. Moreover, of the studies that examine the mental health effects of vicarious exposure to discrimination, few investigate this relationship from a stress and coping perspective beyond the life stages of childhood and adolescence. Thus, the purpose of this study was to assess the effects of vicarious and personal experiences of discrimination on the subjective well-being of Black adults, while observing the potentially moderating effects of utilizing mental health care., Methods: A subsample of Black adults (N = 627) between the ages of 22-69 years old were drawn from the Nashville Stress and Health Study and analyzed to assess within-group variation. Multivariate linear regression was employed to examine the association between vicarious experiences of major discrimination and self-reported anxiety symptoms. Additionally, we evaluated the moderating effects of lifetime utilization of mental health services on the relationship between discrimination and symptoms of anxiety., Results: Findings revealed that vicarious experiences of major discrimination and personal experiences of everyday discrimination were both associated with higher levels of anxiety symptoms among the participants. Additionally, lifetime utilization of mental health care moderated the effects of vicarious and personal experiences of discrimination., Conclusions: The secondhand consequences of discrimination must be considered while assessing the racism-related stress experience. Results from this investigation suggest that mental health treatment should be included in programs targeted to reduce the negative effects of discrimination among Black adults. Additionally, culturally-specific strategies should be considered for addressing racism-related adversity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Racism-related stress frameworks posit that the discriminatory experiences of one's loved ones may threaten one's well-being, but relatively few studies have examined how they may impact mental health beyond childhood and adolescence. Using data from the Nashville Stress and Health Study ( N = 1,252), the present study assessed the prevalence of vicarious experiences of discrimination among subsamples of Black men ( n = 297) and women ( n = 330), examined the association between vicarious experiences of discrimination and psychological distress among Black men and women, and evaluated the impact of vicarious discrimination on psychological distress in the context of other stressors. Results suggest that Black women report more vicarious exposure to specific types of discrimination. Furthermore, vicariously experienced discrimination was associated with higher levels of psychological distress among Black women, but not among Black men. Our findings extend the literature on racism-related stress and offer new insights for interventions aimed at reducing racial disparities in mental health.

Extant literature posits that humans experience two types of threat: physical threat and social threat. While describing pain as "physical" or "social" can be helpful for understanding pain origins (i.e., broken bone versus lost relationship), this dichotomy is largely artificial and not particularly helpful for understanding how the human brain experiences pain. One real world example of social exclusion and rejection that is threatening and likely to bring about significant stress is racism. Racism is a system of beliefs, practices, and policies that operates to disadvantage racialized minorities while providing advantage to those with historical power, particularly White people in the United States and most other Western nations. The objective of this Mini-Review is to present evidence in support of the argument that racism promotes physical pain in racialized minorities, which in turn promotes chronic pain disparities. First, we provide a theoretical framework describing how racism is a potent stressor that affects the health and well-being of racialized minorities. We will then address the neurobiological underpinnings linking racism to social threat, as well as that linking social threats and physical pain. Finally, we will discuss how the perception of social threat brought about by racism may undermine pain management efforts., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Objectives: To evaluate the impact of early life racial discrimination (ELRD) on mental health among Black adults. Methods: Data were from the Nashville Stress and Health Study ( n = 618). Ordinary least squares regression models examined the relationship between ELRD and adult psychological distress; logistic regression estimated the probability of past-year major depressive disorder (MDD). We also assessed whether ELRD moderated the relationship between adult discrimination and mental health. Results: Childhood (b = 1.07, SE = .51, p = .04) and adolescent ELRD (b = 1.32, SE = .42, p = .002) were associated with adult distress. Individuals who experienced childhood ERLD had 88% lower odds of adult MDD than individuals with no ELRD. Significant interactions showed that childhood and adolescent ELRD was protective against adult discrimination. Discussion: While ELRD importantly shapes distress and MDD among Black adults, patterns vary by outcome. Results indicate that adult distress and MDD develop through cumulative adversity processes that are further influenced by sensitive periods in the life course.

This pilot study evaluated the feasibility of implementing an evidence-based, culturally adapted HIV intervention with substance-using African American men in a prison setting. We recruited 60, soon-to-be released African American male prisoners from a larger study ( N = 211) to be randomly selected for participation in the group-based HIV intervention, Real Men Are Safe-Culturally Adapted (REMAS-CA). Participants who were not selected for participation in the intervention received standard Centers for Disease Control and Prevention HIV prevention counseling education, provided during participant assessment in the larger study. Nearly all of the participants who were selected to participate (87%) completed the REMAS-CA intervention. To examine feasibility, data were collected about any revisions made to the HIV intervention components for the prison setting, time needed to implement the program in full, details of implementing the intervention to ensure participation and maintain retention, and participant perception of the intervention. Revisions to the intervention included reducing the number of sessions from five to three, modifying the protocol language based on the setting, and removing six activities deemed inappropriate and/or unallowable by the prison officials. The cultural considerations and intervention, overall, were well received by the participants. However, several revisions made specific to the prison and its administration could affect the potential effectiveness of the intervention. Future research is needed to determine the effectiveness of REMAS-CA among this community.

Objective: The purpose of this study was to assess the utility of the life course framework concept of "linked lives" for examining the effects of partner stress on self-rated health among older adult populations., Method: Data were derived from a partner-dyad study of Miami-Dade County residents and their significant others. We limited our analysis to respondents ages 60 or older (n = 409)., Results: Regression analyses revealed that greater levels of personally experienced major life events were associated with worse self-rated health. However, the association between a significant other's stress exposure and one's own self-rated health was only statistically significant among Black respondents., Discussion: Extending prior study indicating that Black Americans tend to have worse self-rated health later in life relative to other racial groups, these findings demonstrate the utility of the linked lives concept for furthering an understanding of racial disparities in health based upon loved ones' stressful experiences.

Objectives: This study examined whether race/ethnic variation in discrimination is differentially associated with economic adversity during the period of the Great Recession for Blacks and Latinos compared to non-Hispanic Whites, thereby contributing to higher rates of alcohol use and problematic drinking among these groups., Methods: Structural equation modeling was used to analyze data from a national mail survey., Results: The association of 9/11-related discrimination with problem-related drinking substantially derives from the association between 9/11-related discrimination and recession-era economic adversity. The association between 9/11-related discrimination and economic adversity is also significantly greater for Blacks and Latinos compared to non-Hispanic Whites, and is more strongly linked with problem-related drinking for both groups in contrast to non-Hispanic Whites., Conclusions: Overall, the results demonstrate the sustained association of 9/11-related discrimination with alcohol use for Blacks and Latinos compared to non-Hispanic Whites, as well as the differential impact of the Great Recession for these race/ethnic groups. The findings highlight the need to acknowledge macro-level stressors that disproportionately affect disadvantaged groups, such as those occasioned by discriminatory legislation and social policies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Prior research indicates that attention-deficit/hyperactivity disorder (ADHD) is underdiagnosed, or less likely to be diagnosed, among Black children relative to White children and children in other non-Black racial categories. Scholars have suggested that this may be a result of cultural biases or misconceptions that affect the ways classroom behavior is interpreted. The purpose of this pilot study was to engage a larger theoretical framework that explores the relationships between parents and teachers and to examine some of the ways in which common cultural misconceptions can lead to flawed behavioral ascriptions in the classroom, producing negative social outcomes for Black children. Findings from ethnography and interviews reveal that the most common barriers in this low-income neighborhood school setting included poor parent-teacher rapport, a general lack of basic understanding for how ADHD can affect classroom behavior, and faulty procedures in the school setting based on cultural stereotypes. These findings suggest that school officials' disinclination to recommend ADHD testing for Black children may be largely a result of the aforementioned obstacles. A larger study based on these results may produce more robust findings about the barriers that contribute to racial disparities in ADHD diagnoses.

Alpha-Fetoprotein (AFP) is a 68 kDa glycoprotein expressed at high levels by the fetal liver and yolk with transcription repressed to very low levels after birth. Transfer of fetal AFP through the placenta into the circulation of the mother is correlated with remission of rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders. AFP is therefore under development as a biopharmaceutical for the treatment of autoimmune diseases. The clinical evaluation of AFP requires the production of hundreds of grams of highly purified and biologically active protein. We have produced goats that express a form of the human AFP transgene under the control of the beta-casein promoter. In this form of rhAFP, the single N-linked glycosylation site was removed by mutagenesis (N233Q). Here, we describe a purification protocol for this recombinant human (rh)AFP from the milk of these transgenic goats. A three-column procedure was developed to produce gram quantities of highly purified rhAFP. Near- and far-UV circular dichroism spectra of human umbilical cord blood AFP and rhAFP were essentially identical, suggesting that the structure is not affected by removal of the glycosylation site. Furthermore, the cell binding and pharmacokinetics of purified rhAFP were similar to human AFP isolated from cord blood. Our results demonstrate that an active form of rhAFP can be produced on industrial scale by expression in transgenic goat milk.

Dendritic cells (DC) are potent inducers of acquired immunity due to their ability to present antigens in the context of a costimulatory environment and consequently serve an essential role in vaccine efficacy. Strategies to enhance their function, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 treatment to induce DC differentiation from peripheral blood monocytes, may therefore be useful as vaccine adjuvants. We now have evaluated the effect of recombinant GM-CSF on the differentiation of DC in swine. GM-CSF mRNA was readily detected in porcine splenocytes, with increased levels following treatment of the cells with ConA and LPS. Porcine GM-CSF was cloned and expressed in the methylotrophic yeast, Pichia pastoris, as a glycosylated protein that induced proliferation of porcine bone marrow cells. P. pastoris-derived GM-CSF induced expression of antigen presenting (MHC class II) and costimulatory (CD80-CD86) molecules and enhanced antigen presenting cell (APC) function consistent with the induction of functional DC. Thus, recombinant GM-CSF produced by P. pastoris may be a potent adjuvant for swine vaccines., (Copyright 2003 Elsevier Science B.V.)

In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.

Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.

A Pichia pastoris expression clone has been developed to produce the human cytokine interleukin-17 (hIL-17). Characterization of purified recombinant hIL-17 made with this clone demonstrated that it shared many characteristics with hIL-17 produced in mammalian cells. The hIL-17 produced in Pichia had the correct N-terminus of natural mature hIL-17 and a glycosylation pattern similar to hIL-17 produced in mammalian cells; both Pichia and human cells add approximately 5 kDa of sugars via N-linked glycosylation and both express a mixture of the glycosylated and nonglycosylated forms. Gel filtration provides evidence that the Pichia produced hIL-17 exists as a dimer in solution. A combination of cation-exchange and gel-filtration chromatography yielded 3.5 mg of highly purified and biologically active hIL-17 from a 10-liter fermentation. These results show that P. pastoris is a useful system to produce recombinant hIL-17 in structure/function studies of this molecule.

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Q beta replicase can replicate a single molecule of certain species of RNA to 10(14) copies in minutes. This replication ability has been used for in vitro studies of molecular evolution and is currently being utilized as a method of amplifying RNAs that contain probe sequences. It has been observed that Q beta replicase can produce replicatable RNA even in the absence of exogenously added template RNA. The origin of this RNA has been ascribed either to contamination with replicatable RNA or to an ability of Q beta replicase to synthesize RNA de novo from the nucleotides present in the reaction. Technologies that employ Q beta replicase require a thorough understanding of the conditions that lead to this so-called spontaneous RNA production. We have created an expression system and purification method with which we produce gram quantities of highly purified Q beta replicase, and we have identified reaction conditions that prevent the amplification of RNA in assays that do not contain added RNA. However, when these reaction conditions are relaxed, spontaneous RNA replication is seen in up to 100% of the assays. To understand the origin of this RNA, we have cloned several spontaneously produced RNAs. Sequence analysis of one of these RNAs shows that it arose by the evolution of Escherichia coli tRNA into a replicatable template and not by de novo synthesis from nucleoside triphosphates in the reaction.

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.

When cultured cells are injected with mixtures of cores of two reovirus strains, a high proportion of reassortants are monoreassortants, that is, virus particles that contain one genome segment of 1 parent and 9 genome segments of the other. We have isolated two complete sets of monoreassortants, those that contain a single serotype 2 genome segment and 9 serotype 3 genome segments, and those that contain 1 serotype 3 genome segment and 9 serotype 1 genome segments. We have used the former set of monoreassortants (because reovirus serotypes 2 and 3 are less closely related than serotypes 1 and 3) to assess the effect of all 10 genome segments, or rather of the proteins that they encode, in controlling parameters of the reovirus multiplication cycle such as yield size, extent of viral ssRNA, dsRNA and protein synthesis, plaque size, and cytopathogenicity. Among the major findings are: proteins lambda 2, mu 1/mu 1C, and sigma 3 control yield size and extent of RNA and protein synthesis; proteins mu 2 and sigma 1 control severity of cytopathic effects; and proteins sigma 1, mu 1/mu 1C, and mu 2 control plaque size. Identification of monoreassortant phenotypes is useful for identifying which viral proteins are functionally involved at the various stages of the reovirus multiplication cycle.

The uptake of iron supplied as ferric citrate or ferric parabactin was examined in aerobically grown whole cells and vesicles of Rhodopseudomonas sphaeroides. Inner and outer membrane fractions from R. sphaeroides contained no membrane proteins which were inducible by growth in low-iron medium. Vesicles composed of the inner membrane and devoid of outer membrane and periplasmic proteins were able to transport iron supplied as ferric citrate and ferric parabactin. This uptake required the presence of NADH. When the electrical component of the proton motive force was depleted in whole cells, the uptake of iron supplied as ferric parabactin was completely inhibited. The uptake of iron supplied as ferric citrate was inhibited by gallium citrate; however, Ga3+ was not transported. The relationship between iron uptake and heme synthesis was examined by treating whole cells with N-methylprotoporphyrin which inhibits ferrochelatase, the enzyme which inserts ferrous iron into protoporphyrin to form heme. This treatment reduced ferrochelatase activity by 82% but had no effect on iron uptake, indicating that iron uptake and heme synthesis are not directly coupled. The fate of transported iron was investigated by measuring intracellular concentrations of heme and nonheme iron. It was determined that newly transported iron exists primarily as nonheme iron.

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.

The improved Streptex method for serogrouping streptococci incorporates a new extraction enzyme and a simplified procedure requiring no centrifugation. A total of 114 clinical isolates of beta-hemolytic streptococci were serogrouped from primary plates, isolation plates, and Todd-Hewitt broth cultures using this system. Results were compared to those of the heat extraction Lancefield precipitin method. An additional 33 stock culture isolates of related streptococcal species and 5 strains of Listeria were serogrouped to assess the specificity of the test. Agreement between the two methods was 82.5% with primary plates and 96.5% with both isolation plates and broth cultures. Four isolates from three different serogroups were nongroupable by the Lancefield method, but did agglutinate in specific Streptex antisera; therefore, the enzyme extraction procedure appeared more sensitive than the heat extraction method. Streptex accurately grouped five isolates of gamma-hemolytic group B streptococci, but failed to detect antigen in 33% of the group D streptococcal extracts tested. In addition, cross-reactions were observed with strains of alpha-hemolytic streptococci. Streptex produced fewer ambiguous results and required fewer repeat tests. When used with isolation plates or broth cultures, Streptex is both sensitive and specific for the grouping of beta-hemolytic streptococci of groups A, B, C, F, and G.

Identification of viral genes that specify virulence, however defined, is of critical importance for the design of viral vaccines. In particular, the targeted development not only of avirulent vaccine strains but also of viruses to be used as carriers for foreign genes depends on the inactivation or deletion of genes that harm the host. This paper illustrates approaches to identifying viral pathogenesis genes in two model systems involving cowpox virus and reovirus, respectively.

Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media. The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively. Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate. Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques. The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.

A direct fluorometric test for the rapid detection of group A streptococci from throat swabs was compared with the microscopic fluorescent-antibody test. Formalinized throat swab cultures (490) were examined by the two methods, and the results agreed on 84% of the specimens. In another comparison, 15-hr broth cultures of 103 freshly taken throat swabs were tested by both methods. Of the specimens tested, 101 (98%) were either positive or negative by both methods. In all cases, the latter results correlated with the demonstration of presence or absence of group A streptococci in the specimens by cultural isolation and precipitin grouping tests. It may be feasible to use the direct fluorometric test in a diagnostic laboratory as described or possibly to adapt it for automatic processing of throat swab cultures.