Conditions of successful treatment referral practices with justice-involved youth: Qualitative insights from probation and service provider staff involved in JJ-TRIALS.

INTRODUCTION: Compared to the general U.S. adolescent population, young people involved in the juvenile justice system are at greater risk of experiencing substance use (SU) issues. There are critical opportunities across the juvenile justice continuum, at points of interface with community-based treatment services, to screen and assess for SU issues, identify unmet treatment needs, and refer those in need to treatment. The treatment referral process is, however, complex, and contingent on a seamless nexus between juvenile justice operations and the wider treatment provider landscape. Given the lack of successful SU referrals among justice-involved youth and the variable referral rates across jurisdictions, this study's aim is to provide a qualitative, explanatory understanding of the conditions that together contribute to successful referring practices. METHODS: The study is based on an analysis of a qualitative dataset comprising focus group data with probation and community-based behavioral health treatment staff working in 31 sites in 6 different states as part of the clustered randomized trial of an organizational change intervention known as JJ-TRIALS (Juvenile Justice Translational Research on Interventions for Adolescents in the Legal System). The data contain respondents' narratives on the achievements, successes, and challenges with implementing the intervention. The data were analyzed through a combination of strategies to identify the conditions that both facilitate and impede referral processes between probation offices and community-based SU treatment providers. RESULTS: Participants across sites discussed the positive impacts that the JJ-TRIALS intervention had on their improved ability to communicate, collaborate, and collect data. From the interviews, seven main conditions were observed to contribute to successful SU treatment referral practices: (1) communication (inter-organizational); (2) collaboration; (3) data-driven practices; (4) family engagement; (5) institutionalized policy and referral documentation; (6) efficient referral policies and procedures; and (7) suitable and accessible system of treatment providers. CONCLUSION: Findings highlight the value of a holistic understanding of successful treatment referrals for justice-involved youth and help inform research and practice efforts to identify and measure the many dimensions of referral-making at the interface of juvenile probation and behavioral health services.

A multiplexed barcode approach to simultaneously evaluate gene delivery by adeno-associated virus capsid variants in nonhuman primates.

BACKGROUND AND AIMS: Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs. APPROACH AND RESULTS: Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal's distinctive serological status. CONCLUSIONS: This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type.

Contexts shaping misdemeanor system interventions among people with mental illnesses: qualitative findings from a multi-site system mapping exercise.

BACKGROUND: People with mental illnesses are disproportionately entangled in the criminal legal system. Historically, this involvement has resulted from minor offending, often accompanied by misdemeanor charges. In recent years, policymakers have worked to reduce the footprint of the criminal legal system. This paper seeks to better understand how misdemeanor systems intervene in the lives of people with mental illnesses. METHODS: System mapping exercises were conducted with misdemeanor system stakeholders from the jurisdictions of Atlanta, Chicago, Manhattan, and Philadelphia. Narrative detail on decision-making and case processing, both generally and in relation to specific types of behavior, including trespassing, retail theft/shoplifting, and simple assault, were coded and analyzed for thematic patterns. Based on the qualitative analysis, this paper offers a conceptual diagram of contexts shaping misdemeanor system interventions among people with mental illnesses. RESULTS: All four sites have been engaged in efforts to reduce the use of misdemeanor charges both generally and in relation to people with mental illnesses. Decision-makers across all sites experience contexts that shape how, when, and where they intervene, which are: (1) law and policy environments; (2) location of the behavior; (3) expectations of stakeholders; (4) knowledge of mental illnesses; and (5) access to community resources. Law and policy environments expand or constrain opportunities for diversion. The location of offending is relevant to who has a stake in the behavior, and what demands they have. Clinical, experiential, and system-level knowledge of mental illnesses inform a chain of decisions about what to do. The capacity to address mental health needs is contingent on access to social services, including housing. CONCLUSION: People making decisions along the criminal legal continuum are critical to illuminating the dynamic, inter-related contexts that facilitate and frustrate attempts to address defendants' mental health needs while balancing considerations of public safety. Multi-sector, scenario-based or case study exercises could help identify concrete ways of improving each of the contexts that surround whole-of-system decisions.

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Nanoparticle Biokinetics in Mice and Nonhuman Primates.

Despite the preponderance of iron oxide nanoparticles (NPs) designed for theranostic applications, widespread clinical translation of these NPs lags behind. A better understanding of how NP pharmacokinetics vary between small and large animal models is needed to rapidly customize NPs for optimal performance in humans. Here we use noninvasive magnetic resonance imaging (MRI) to track iron oxide NPs through a large number of organ systems in vivo to investigate NP biokinetics in both mice and nonhuman primates. We demonstrate that pharmacokinetics are similar between mice and macaques in the blood, liver, spleen, and muscle, but differ in the kidneys, brain, and bone marrow. Our study also demonstrates that full-body MRI is practical, rapid, and cost-effective for tracking NPs noninvasively with high spatiotemporal resolution. Our techniques using a nonhuman primate model may provide a platform for testing a range of NP formulations.

No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model.

Umbilical cord blood transplant continues to increase in prevalence as a treatment option for various hematopoietic and immune disorders. Because of the limited number of cells available in a single cord blood unit, investigators have explored methods of increasing cell dose before transplant, including overexpression of the homeobox B4 (HOXB4) transcription factor. We have previously reported the development of leukemia in several nonhuman primate (NHP) subjects transplanted with HOXB4-expanded bone marrow cells at approximately 2 years posttransplant. Here, we provide long-term data for a NHP receiving a HOXB4-expanded cord blood graft. Longitudinal follow-up included gene marking analysis, complete blood counts, morphologic/pathologic assessment, phenotypic analysis of subsets, and retroviral integration site analysis. In each of these independent assays, we saw no indication of clonal dominance, and all signs pointed toward normal, healthy hematopoiesis. Furthermore, in-depth clonal analysis of an animal that developed leukemia after transplantation of HOXB4-modified bone marrow cells showed that dominant clones could be detected as early as 6 months posttransplant using the genomic analysis technique detailed here. Parallel analysis of the cord blood transplant macaque showed no such sites. These findings demonstrate the ability to study the use of gene-modified and expanded cord blood cells in a NHP model.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Transmission of Chagas disease via blood transfusions in 2 immunosuppressed pigtailed macaques (Macaca nemestrina).

A 2.25-y-old male pigtailed macaque (Macaca nemestrina) was experimentally irradiated and received a bone marrow transplant. After transplantation and engraftment, the macaque had unexpected recurring pancytopenia and dependent edema of the prepuce, scrotum, and legs. The diagnostic work-up included a blood smear, which revealed a trypomastigote consistent with Trypanosoma cruzi, the causative agent of Chagas disease (CD). We initially hypothesized that the macaque had acquired the infection when it lived in Georgia. However, because the animal had received multiple blood transfusions, all blood donors were screened for CD. One male pigtailed macaque blood donor, which was previously housed in Louisiana, was positive for T. cruzi antibodies via serology. Due to the low prevalence of infection in Georgia, the blood transfusion was hypothesized to be the source of T. cruzi infection. The transfusion was confirmed as the mechanism of transmission when screening of archived serum revealed seroconversion after blood transfusion from the seropositive blood donor. The macaque made a full clinical recovery, and further follow-up including thoracic radiography, echocardiography, and gross necropsy did not show any abnormalities associated with CD. Other animals that received blood transfusions from the positive blood donor were tested, and one additional pigtailed macaque on the same research protocol was positive for T. cruzi. Although CD has been reported to occur in many nonhuman primate species, especially pigtailed macaques, the transmission of CD via blood transfusion in nonhuman primates has not been reported previously.

Ectopic expression of HOXC6 blocks myeloid differentiation and predisposes to malignant transformation.

Insertional mutagenesis resulting from the integration of retroviral vectors has led to the discovery of many oncogenes associated with leukemia. We investigated the role of HOXC6, identified by proximal provirus integration in a large animal hematopoietic stem cell gene therapy study, for a potential involvement in hematopoietic stem cell activity and hematopoietic cell fate decision. HOXC6 was overexpressed in the murine bone marrow transplantation model and tested in a competitive repopulation assay in comparison to the known hematopoietic stem cell expansion factor, HOXB4. We have identified HOXC6 as a factor that enhances competitive repopulation capacity in vivo and colony formation in vitro. Ectopic HOXC6 expression also induced strong myeloid differentiation and expansion of granulocyte-macrophage progenitors/common myeloid progenitors (GMPs/CMPs) in vivo, resulting in myeloid malignancies with low penetrance (3 of 17 mice), likely in collaboration with Meis1 because of a provirus integration mapped to the 3' region in the malignant clone. We characterized the molecular basis of HOXC6-induced myeloid differentiation and malignant cell transformation with complementary DNA microarray analysis. Overexpression of HOXC6 induced a gene expression signature similar to several acute myeloid leukemia subtypes when compared with normal GMPs/CMPs. These results demonstrate that HOXC6 acts as a regulator in hematopoiesis and is involved in malignant transformation.

CD34(+) expansion with Delta-1 and HOXB4 promotes rapid engraftment and transfusion independence in a Macaca nemestrina cord blood transplant model.

Umbilical cord blood (CB) transplantation is a promising therapeutic approach but continues to be associated with delayed engraftment and infections. Here, we explored in our macaque CB transplant model expansion and engraftment kinetics of cells expanded with the combination of HOXB4 and Delta-1. CB cells were divided into two equal fractions; one fraction was transduced with HOXB4 yellow fluorescent protein (YFP) and expanded on control OP9 cells, and the other was transduced with HOXB4 green fluorescent protein (GFP) and expanded on Delta-expressing OP9 cells (OP9-DL1). Both fractions were transplanted into myeloablated subjects. Neutrophil and platelet recovery occurred within 7 and 19 days respectively, which was significantly earlier than in our previous study using cells expanded with HOXB4 alone, which resulted in neutrophil recovery within 12 days (P = 0.05) and platelet recovery within 37 days (P = 0.02). Furthermore, two of three animals in the current study remained fully transfusion-independent after transplantation. By day 30, reconstitution of lymphocytes was significantly greater with the HOXB4/OP9-DL1 expanded cells in all animals (P = 0.05). In conclusion, our data show that the combination of OP9-DL1 and HOXB4 can result in increased numbers of repopulating cells, thus leading to rapid engraftment and transfusion independence in macaques transplanted with autologous, expanded CB cells.

Hematopoietic stem cell expansion facilitates multilineage engraftment in a nonhuman primate cord blood transplantation model.

The use of umbilical cord blood for allogeneic transplantation has increased dramatically over the past years. However, the limited number of cells available in a single cord blood unit remains a serious obstacle. Here, we wished to establish a nonhuman primate cord blood transplantation model that would allow us to test various hematopoietic stem cell expansion and gene therapy strategies. We implemented HOXB4-mediated expansion based on our previous experience with HOXB4 in autologous cells. Cord blood units were divided into two equal parts; half of the cells were transduced with a yellow fluorescent protein control vector and cryopreserved, and half were transduced with a HOXB4GFP vector, expanded, and cryopreserved. Both fractions of cells were transplanted into Macaca nemestrina subjects. We found that neutrophil recovery occurred within 19 days in all animals, and both neutrophil and platelet recovery were substantially accelerated compared to human single unit cord blood transplants. In addition, HOXB4-transduced and expanded cells resulted in superior engraftment of all hematopoietic lineages in all animals over nonexpanded controls. In conclusion, we have successfully established a nonhuman primate cord blood transplantation model and demonstrated that HOXB4 stimulates expansion and engraftment of repopulating cells. The availability of such a model has significant implications for developing and testing strategies to improve clinical cord blood transplantation, as it will allow comparison of different stem cell expansion methodologies within a single animal. Furthermore, it can be used in long-term follow-up studies to determine how specific expansion techniques affect engraftment of various hematopoietic lineages.

Teaching phoneme awareness to pre-literate children with speech disorder: a randomized controlled trial.

BACKGROUND: Awareness of individual phonemes in words is a late-acquired level of phonological awareness that usually develops in the early school years. It is generally agreed to have a close relationship with early literacy development, but its role in speech change is less well understood. Speech and language therapy for children with speech disorder involves tasks that appear, either implicitly or explicitly, to require a phonemic level of awareness. However, children typically attend for intervention at a pre-school, pre-literate stage, i.e. before they would be expected to have developed the relevant phoneme segmentation and manipulation skills. AIMS: To investigate whether it is possible to teach phoneme awareness skills to pre-literate children with speech disorder. METHODS & PROCEDURES: In a randomized controlled trial design 42 children with speech disorder, aged 4;0-4;6, were allocated to either a phonological awareness or a language stimulation programme. Children were assessed on four measures of phoneme awareness (alliteration awareness, phoneme isolation, word segmentation and phoneme addition/deletion) immediately before and after the programme and categorized as 'improved' or 'not improved' according to predetermined criteria. Fisher's Exact test was used to compare outcome in the two groups. OUTCOMES & RESULTS: Significantly more children improved in the phonological awareness group than in the language stimulation group for three out of the four measures (all except alliteration awareness). However, for the two most advanced tasks (segmentation and addition/deletion) only a small minority of children showed improvement. A marked improvement in Phoneme Isolation was made by the majority of children in the phonological awareness group. CONCLUSIONS: It is possible to teach some advanced phoneme awareness skills to some pre-literate children. Phoneme Isolation was the most easily learned and is a skill that appears very relevant to speech and language therapy. However, phoneme addition, deletion and word segmentation showed relatively limited improvement and only in a small number of cognitively able and older children. Whereas isolation of word initial consonants appears to be a skill that can be triggered at 4;0-4;6 by relevant activities, most children in the study were not cognitively ready for more advanced, abstract phoneme manipulation tasks. This raises questions about how speech and language therapists should tackle many common errors and the age at which we should aim to develop or draw on phoneme awareness to stimulate speech change.