Adolescent health and well-being in sub-Saharan Africa: Strengthening knowledge base and research capacity through a collaborative multi-country school-based study.

In Sub-Saharan Africa (SSA), adolescents make up around one-quarter of the population who are growing up in a rapidly urbanizing environment, with its associated risks and benefits, including impacts on health, psychosocial development, nutrition, and education. However, research on adolescents' health and well-being in SSA is limited. The ARISE (African Research, Implementation Science and Education) Network's Adolescent Health and Nutrition Study is an exploratory, school-based study of 4988 urban adolescents from five countries: Burkina Faso, Ethiopia, South Africa, Sudan, and Tanzania. A multistage random sampling strategy was used to select the schools and adolescents. Adolescent boys and girls aged 10-15 years were interviewed using a standardized questionnaire by trained enumerators. The questionnaire covered multiple domains including demographic and socioeconomic characteristics, water, sanitation and hygiene practices, antimicrobial resistance, physical activity, dietary behaviours, socioemotional development, educational outcomes, media use, mental health, and menstrual hygiene (only for girls). Additionally, a desk review of health and school meal policies and programs and a qualitative investigation into health and food environments in schools were conducted with students, administrators, and food vendors. In this paper, we describe the study design and questionnaire, present profiles of young adolescents who participated in the study, and share field experiences and lessons learned for future studies. We expect that this study along with other ARISE Network projects will be a first step toward understanding young people's health risks and disease burdens, identifying opportunities for interventions and improving policies, as well as developing potential research capacities on adolescent health and well-being in the SSA region.

A Bispecific METxMET Antibody-Drug Conjugate with Cleavable Linker Is Processed in Recycling and Late Endosomes.

Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

Meals, Education, and Gardens for In-School Adolescents (MEGA): study protocol for a cluster randomised trial of an integrated adolescent nutrition intervention in Dodoma, Tanzania.

INTRODUCTION: Secondary schools have the transformative potential to advance adolescent nutrition and provide a unique entry point for nutrition interventions to reach adolescents and their families and communities. Integrated school nutrition interventions offer promising pathways towards improving adolescent nutrition status, food security and building sustainable skill sets. METHODS AND ANALYSIS: The Meals, Education, and Gardens for In-School Adolescents (MEGA) project aims to implement and evaluate an integrated, school-based nutrition intervention package among secondary schools in the Chamwino District of Dodoma, Tanzania. MEGA is a cluster-randomised controlled trial, including six public secondary schools assigned to three different arms. Two schools will receive the full intervention package, including school meals, school gardens, nutrition education and community workshops. Two schools will receive the partial intervention package, including the school garden, nutrition education and community workshops. Two schools will serve as the controls and will not receive any intervention. The intervention will be implemented for one academic year. Baseline and end-line quantitative data collection will include 750 adolescents and 750 parents. The domains of outcomes for adolescents will include haemoglobin concentrations, anthropometry, educational outcomes and knowledge, attitudes and practices regarding nutrition, agriculture and health. The domains of outcomes for parents will include knowledge, attitudes and practices of nutrition, agriculture and health. End-line focus group discussions will be conducted among selected adolescents, parents and teachers to assess the facilitators and barriers associated with the intervention. ETHICS AND DISSEMINATION: This study was approved by the Institutional Review Board at Harvard T.H. Chan School of Public Health (approval number: IRB20-1623), the Institutional Research Review Committee at the University of Dodoma (approval number: MA.84/261/02) and the Tanzania National Institute for Medical Research (approval number: NIMR/HO/R.8a/Vol. IX/3801). A manuscript with the research findings will be developed for publication. Local dissemination meetings will be held with key stakeholders. TRIAL REGISTRATION NUMBER: NCT04788303.; ClinicalTrials.gov Identifier.

Disruption of nuclear envelope integrity as a possible initiating event in tauopathies.

The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.

Impacts of school feeding on educational and health outcomes of school-age children and adolescents in low- and middle-income countries: A systematic review and meta-analysis.

BACKGROUND: School feeding programs are ubiquitous in low- and middle-income countries (LMICs) and may have critical implications for the health and education of school-age children and adolescents. This systematic review aimed to assess the impacts of school feeding on educational and health outcomes of children and adolescents in LMICs. METHODS: Interventional studies on the effects of school feeding on nutritional and health outcomes of children and adolescents receiving primary or secondary education in LMICs were included. MEDLINE, EMBASE, CINAHL, the Cochrane Library, and grey literature were searched (through December 2019) to identify eligible studies. We included randomized controlled trials and controlled before-after studies on school feeding conducted in LMICs among children and adolescents aged 6 to 19 who received primary or secondary education. Two reviewers independently conducted study selection, data extraction, and risk of bias assessment. Meta-analyses were performed for outcomes available in three or more independent studies. Subgroup analyses were conducted by study design and school feeding modality whenever possible. RESULTS: Fifty-seven articles met the inclusion criteria for the review, including 44 randomized controlled trials and 13 controlled before-after studies; 19 articles were included in the meta-analysis. School feeding resulted in a significant increase in height (mean difference = 0.32 cm; confidence interval (CI) = 0.03, 0.61; P = 0.032) and weight (mean difference: 0.58 kg; 95% 95% CI = 0.22, 0.93; P = 0.001) over 12 months, compared to those in the control groups. School feeding also resulted in a significant increase in the percentage of school days attended (2.6%; 95% CI = 1.2%, 3.9%; P < 0.001). CONCLUSIONS: School feeding is an important approach to improving the health and education outcomes of children and adolescents living in LMICs. More well-designed research is needed to establish further the effectiveness of school feeding for nutritional outcomes and academic achievement. REGISTRATION: PROSPERO ID: CRD42020159003.

School-Based Nutrition Programs for Adolescents in Dodoma, Tanzania: A Situation Analysis.

BACKGROUND: Tanzania has a double burden of malnutrition, including a high prevalence of undernutrition and an increasing prevalence of overweight and obesity among adolescents. Schools present a valuable opportunity to reach a large section of the country's adolescent population with nutrition-oriented interventions. OBJECTIVE: The objective of this study was to assess the current state of adolescent school nutrition interventions in Dodoma, Tanzania, with emphasis on 3 potential school-based nutrition interventions, school vegetable gardens, school meals, and education (on nutrition, agriculture, and water, sanitation, and hygiene). METHODS: Focus group discussions were conducted with several regional and district-level governmental stakeholders, including health, education, and agricultural officers. Ten public secondary schools were visited, and interviews with school administrators, teachers, students, and parents were conducted. RESULTS: All stakeholders interviewed supported interventions to improve school-based nutrition, including school gardens, school feeding, and nutrition education. All 10 schools visited had some experience providing school meals, but parents' contributions were essential for the program's sustainability. Most schools visited had land available for a school garden program, but water availability could be challenging during certain times of the year. The teachers interviewed expressed that the curriculum on nutrition education was highly theoretical and did not allow students to practice the knowledge and skills they learned in the classroom. CONCLUSIONS: The current school-based approach to tackling the double burden of adolescent malnutrition in Dodoma is localized and ad hoc. To leverage the potential of schools as a platform for nutrition interventions, integrated and policy-mandated interventions are needed.

The COVID-19 Pandemic and Adolescents' Experience in Sub-Saharan Africa: A Cross-Country Study Using a Telephone Survey.

The public health measures instituted by governments to combat the coronavirus disease 2019 (COVID-19) may cause developmental and educational losses to adolescents. The impacts of the COVID-19 pandemic and its mitigation strategies on adolescents in sub-Saharan Africa are unclear. This study aimed to examine adolescents' knowledge, perceptions, and practices related to COVID-19 and the impacts of the pandemic on the daily lives of adolescents in sub-Saharan Africa. The survey was conducted in Burkina Faso, Ethiopia, and Nigeria using computer-assisted telephone interviews to enable rapid and remote data collection. Two sites were included in each country, with approximately 300 adolescents per site and 1,795 adolescents in total. Variations across the six sites were noted for the proportions of the adolescents who could correctly identify all key COVID-19 symptoms (4-25%), transmission methods (16-59%), and prevention approaches (33-79%). Most (> 72%) of the adolescents were no longer going to school due to school closures. Many adolescents (23-81%) were not receiving any education during the pandemic. A considerable proportion of the adolescents (44-83%) self-assessed as having less ability to learn during the pandemic; many expected it to be very difficult to catch up on education after the pandemic. Decreases in the consumption of major food groups were common across sites. Urgent actions are needed in sub-Saharan Africa to address the inadequate knowledge of COVID-19 among adolescents and the impacts of the pandemic on adolescent education and nutrition.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

Autophagy protects tumors from T cell-mediated cytotoxicity via inhibition of TNFα-induced apoptosis.

Although T cell checkpoint inhibitors have transformed the treatment of cancer, the molecular determinants of tumor cell sensitivity to T cell-mediated killing need further elucidation. Here, we describe a mouse genome-scale CRISPR knockout screen that identifies tumor cell TNFα signaling as an important component of T cell-induced apoptosis, with NF-κB signaling and autophagy as major protective mechanisms. Knockout of individual autophagy genes sensitized tumor cells to killing by T cells that were activated via specific TCR or by a CD3 bispecific antibody. Conversely, inhibition of mTOR signaling, which results in increased autophagic activity, protected tumor cells from T cell killing. Autophagy functions at a relatively early step in the TNFα signaling pathway, limiting FADD-dependent caspase-8 activation. Genetic inactivation of tumor cell autophagy enhanced the efficacy of immune checkpoint blockade in mouse tumor models. Thus, targeting the protective autophagy pathway might sensitize tumors to T cell-engaging immunotherapies in the clinic.

The 3'UTR of HIC mRNA improves the production of recombinant proteins in Chinese hamster ovary cells.

Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70-80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3' untranslated region (3'UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3'UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3'UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-gamma in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3'UTR in CHO cells.

Genome-scale CRISPR activation screen uncovers tumor-intrinsic modulators of CD3 bispecific antibody efficacy.

Bispecific antibodies (bsAb) that bridge tumor cells and CD3-positive effector T cells are being developed against many tumor cell targets. While tumor cell factors other than target expression level appear to play a role in determining the efficacy of CD3 bsAb, the identity of such factors remains largely unknown. Using a co-culture system of primary human T cells and B lymphoma cell lines, we demonstrate a range of sensitivities to CD20xCD3 bsAb that is independent of CD20 surface expression. To identify genes that modulate tumor cell sensitivity to CD3 bsAb, we employed a genome-scale CRISPR activation screen in a CD20xCD3-sensitive human B lymphoma cell line. Among the most highly enriched sgRNAs were those targeting genes with predicted effects on cell-cell adhesion, including sialophorin (SPN). Increased expression of SPN impeded tumor cell clustering with T cells, thereby limiting CD3 bsAb-mediated tumor cell lysis. This inhibitory effect of SPN appeared to be dependent on sialylated core 2 O-glycosylation of the protein. While SPN is not endogenously expressed in the majority of B cell lymphomas, it is highly expressed in acute myeloid leukemia. CRISPR-mediated SPN knockout in AML cell lines facilitated T cell-tumor cell clustering and enhanced CD3 bsAb-mediated AML cell lysis. In sum, our data establish that the cell cross-linking mechanism of CD3 bsAb is susceptible to subversion by anti-adhesive molecules expressed on the tumor cell surface. Further evaluation of anti-adhesive pathways may provide novel biomarkers of clinical response and enable the development of effective combination regimens for this promising therapeutic class.

Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription.

Positive transcription elongation factor b (P-TEFb) complexes, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 or T2, are engaged by many cellular transcription regulators that activate or inhibit transcription from specific promoters. The related I-mfa (inhibitor of MyoD family a) and HIC (human I-mfa-domain-containing) proteins function in myogenic differentiation and embryonic development by participating in the Wnt signaling pathway. We report that I-mfa is a novel regulator of P-TEFb. Both HIC and I-mfa interact through their homologous I-mfa domains with cyclin T1 and T2 at two binding sites. One site is the regulatory histidine-rich domain that interacts with CDK9 substrates including RNA polymerase II. The second site contains a lysine and arginine-rich motif that is highly conserved between the two T cyclins. This site overlaps and includes the previously identified Tat/TAR recognition motif of cyclin T1 required for activation of human immunodeficiency virus type 1 (HIV-1) transcription. HIC and I-mfa can serve as substrates for P-TEFb. Their I-mfa domains also bind the activation domain of HIV-1 Tat and inhibit Tat- and P-TEFb-dependent transcription from the HIV-1 promoter. This transcriptional repression is cell-type specific and can operate via Tat and cyclin T1. Genomic and sequence comparisons indicate that the I-mf and HIC genes, as well as flanking genes, diverged from a duplicated chromosomal region. Our findings link I-mfa and HIC to viral replication, and suggest that P-TEFb is modulated in the Wnt signaling pathway.

Transscleral fine-needle aspiration biopsy of macular choroidal melanoma.

PURPOSE: To report transscleral 30-gauge fine-needle aspiration biopsy (FNAB) for cytology and cytogenetics in eyes with macular choroidal melanoma. DESIGN: Prospective, interventional case series. METHODS: Twenty-five patients (25 eyes) who underwent transscleral 30-gauge FNAB of macular choroidal melanoma immediately prior to iodine-125 plaque placement were included in this study, conducted at a tertiary care university hospital. The main outcome measures were FNAB feasibility, cytology, cytogenetic analysis for monosomy 3, and surgical complications. RESULTS: Transscleral 30-gauge FNAB of choroidal melanoma in the macula was performed in 24 of 25 (96%) eyes and was not feasible owing to insufficient exposure in one eye (4%). Biopsy was diagnostic of choroidal melanoma in 17 of 24 (71%) eyes. Fluorescent in situ hybridization (FISH) and/or GeneChip 500k NspI Mapping array (Affymetrix, Santa Clara, California, USA) analysis for monosomy 3 was completed in 16 of 24 (67%) revealing monosomy 3 in five eyes and disomy 3 in 11 eyes. Retinal perforation (four eyes) did not require treatment or result in retinal detachment; submacular hemorrhage (nine eyes) and vitreous hemorrhage (five eyes) cleared spontaneously within one month. CONCLUSION: Transscleral FNAB of macular choroidal melanoma is feasible in most eyes and frequently yields cytogenetic information relevant to prognosis.

Reduced photoreceptor damage after photodynamic therapy through blockade of nitric oxide synthase in a model of choroidal neovascularization.

PURPOSE: To investigate the role of nitric oxide synthase (NOS) in photoreceptor degeneration associated with photodynamic therapy (PDT) in a laser-induced model of choroidal neovascularization (CNV). METHODS: PDT was performed in monkey and Brown Norway rats with laser-induced CNV. L-NAME, a NOS inhibitor, or saline was injected intraperitoneally in rats with CNV. An NO donor, or saline, was injected intravitreously into normal rats. Photoreceptor apoptosis was evaluated by TUNEL and electron microscopy. NOS, ED-1, and cleaved-caspase-3 (c-casp-3) expression were determined by immunohistochemistry. CNV lesions were examined by fluorescence angiography and choroidal flat mount. RESULTS: TUNEL and electron microscopy showed photoreceptor apoptosis after PDT. In rats, there were significantly more TUNEL-positive cells in the photoreceptors 24 hours after PDT, whereas in the CNV lesions there were more TUNEL-positive cells 6 hours after PDT. C-casp-3 was detected in the CNV lesions but not in the photoreceptors after PDT. There was no difference in the numbers of ED-1-positive macrophages before and after PDT. However, inducible NOS (iNOS) was increased after PDT in macrophages. Intravitreous injection of the NO donor without PDT also induced substantial photoreceptor apoptosis. L-NAME-treated animals had significantly fewer TUNEL-positive cells in the photoreceptors than saline-treated animals after PDT (P < 0.05). There were no differences in CNV size and leakage between L-NAME- and saline-treated groups. CONCLUSIONS: iNOS expression in macrophages contributes to PDT-induced photoreceptor degeneration. NOS inhibition reduces PDT-induced photoreceptor degeneration without compromising the treatment effect of PDT in an experimental model of CNV.

Photodynamic therapy induces caspase-dependent apoptosis in rat CNV model.

PURPOSE: To investigate the mechanism of cell death in laser-induced choroidal neovascularization (CNV) after photodynamic therapy (PDT). METHODS: PDT was performed in Brown-Norway rats using laser light at a wavelength of 689 nm, irradiance of 600 mW/cm(2), and fluence of 25 J/cm(2) after intravenous injection of verteporfin at the doses of 3, 6, and 12 mg/m(2). Apoptotic cells in CNV were detected by TUNEL assay at 1, 3, 6, 15, 24, and 48 hours after PDT. Caspase activation at 1, 3, 6, 15, and 24 hours after PDT was determined by immunohistochemistry (IHC) with a cleaved caspase-3 or -9 antibody. Akt activity was determined by Western blot and IHC with a phosphorylated-Akt (pAkt) antibody. To investigate the roles of Akt in PDT-induced apoptosis, insulin-like growth factor (IGF)-1, an Akt activator, with or without wortmannin, an inhibitor of PI3K-Akt pathway, was injected into the vitreous before PDT. RESULTS: The number of TUNEL-positive cells in CNV increased at 3 hours after PDT and peaked at 6 hours, showing a dose dependence of verteporfin. Caspase activation was detected in TUNEL-positive cells. Dephosphorylation of Akt in CNV occurred within 1 hour. IGF-1 significantly activated Akt and suppressed the number of TUNEL-positive cells in CNV, and the effects of IGF-1 were diminished by wortmannin. CONCLUSIONS: PDT induced caspase-dependent apoptosis in CNV. These results suggest that PDT leads to dephosphorylation of Akt and subsequent activation of the caspase-dependent pathway. Understanding the intracellular signaling mechanisms of apoptosis in PDT may lead to more selective and effective treatment of CNV secondary to age-related macular degeneration.

High-density genome array is superior to fluorescence in-situ hybridization analysis of monosomy 3 in choroidal melanoma fine needle aspiration biopsy.

PURPOSE: Using fluorescence in situ hybridization (FISH) and high-density single nucleotide polymorphism (SNP) mapping genome array, we comparatively evaluated chromosome 3 status and other chromosomal aberrations within a series of choroidal melanomas biopsied by fine needle aspiration (FNAB). METHODS: Transscleral FNAB was performed in 59 patients (59 eyes) who had a clinical diagnosis of choroidal melanoma. Biopsies were processed for chromosome 3 status by centromeric interphase FISH, cytopathology, cell culture, and simultaneous genomic DNA and RNA mapping array analysis. RESULTS: FISH yielded chromosome 3 status in 38 of 59 (64%) eyes, while high-density SNP mapping array yielded chromosome 3 status in 43 of 59 (73%) eyes. Monosomy 3 was detected by FISH in 15 of 38 (39%) cases, and high-density SNP mapping array data confirmed the finding in 13 of the 15 cases. Furthermore, high-density SNP mapping array revealed five additional cases of significant chromosome 3 aberration not detected by FISH. High-density genomic mapping also provided detailed patterns of chromosomal gain and loss on chromosomes 1, 6, 8, and 9 which segregated into two groups characterized by either monosomy 3 or chromosome 6p gain. CONCLUSIONS: High-density SNP mapping array was better than FISH in detecting chromosome 3 aberrations and monosomy in our melanoma samples. More importantly, the mapping arrays detected additional patterns of chromosomal aberration, which suggest specific pathways for cytogenetic rearrangements in choroidal melanoma and may improve prognostic testing.

Fluorescent in situ hybridization for monosomy 3 via 30-gauge fine-needle aspiration biopsy of choroidal melanoma in vivo.

OBJECTIVE: To report the feasibility of intraoperative transscleral fine-needle aspiration biopsy at plaque surgery to obtain cells for monosomy 3 analysis in patients with choroidal melanoma. DESIGN: Consecutive interventional case series. PARTICIPANTS: Eighteen patients (18 eyes) with choroidal melanoma who had fine-needle aspiration biopsy performed with a 30-gauge needle at time of iodine 125 plaque placement. INTERVENTION: Cytology and cytogenetic analysis for monosomy 3 were obtained from biopsy specimens. MAIN OUTCOME MEASURES: Cytology, cytogenetic analysis for monosomy 3, and complications and feasibility of transscleral fine-needle aspiration biopsy of choroidal melanoma in vivo. RESULTS: Fine-needle aspiration biopsy was diagnostic of choroidal melanoma in 14 of 18 cases and resulted in viable cell cultures for fluorescent in situ hybridization (FISH) analysis in 9 cases. Fluorescent in situ hybridization for monosomy 3 was positive in 4 of the 9 cases. One patient had a mild vitreous hemorrhage. Tumors between 2 and 3 mm in height and those that yielded cells that did not attach in culture were most likely to have insufficient growth for FISH analysis. CONCLUSIONS: Transscleral fine-needle aspiration biopsy and FISH for monosomy 3 may provide important prognostic information on patients who undergo plaque radiotherapy for choroidal melanoma.

Surveillance of the eye and vision in clinical trials of CP-675,206 for metastatic melanoma.

PURPOSE: To determine the ocular safety of CP-675,206 (Pfizer, New York, New York, USA), a fully human anti-cytotoxic T lymphocyte-associated antigen 4 monoclonal antibody in clinical trials of immunotherapy of metastatic melanoma. DESIGN: Prospective, nonrandomized study of the eye and vision in phase I/II clinical trials of CP-675,206 in metastatic melanoma conducted at the University of California, Los Angeles. METHODS: Patients with regional or distant metastatic melanoma were enrolled in phase I/II clinical trials evaluating the safety and antitumor efficacy of CP-675,206 alone or in combination with melanoma antigen peptide-pulsed dendritic cell vaccines. Ophthalmic evaluation was performed at the onset of CP-675,206 immunotherapy (baseline evaluation), two months or more after the onset of CP-675,206 immunotherapy (end-study evaluation), and at two- to three-month intervals thereafter in patients who continued to receive CP-675,206 immunotherapy (poststudy evaluation). Baseline and end-study evaluations included comprehensive ophthalmic examination, psychophysical and electrophysiologic visual function assessment, fundus photography, fluorescein angiography, and visual function assessment. RESULTS: Twenty patients with metastatic melanoma arising from the skin, mucosa, eye, or unknown site were evaluated. Systemic toxicity attributed to CP-675,206 included dermatologic manifestations, diarrhea, and autoimmune hepatitis with panhypopituitarism. A subset of patients receiving CP-675,206 demonstrated antitumor efficacy with partial response or complete response of metastatic melanoma. Comparison of ophthalmic baseline with end-study evaluations in all 20 patients and limited-term poststudy evaluations showed no adverse effect of CP-675,206 immunotherapy on the eye or vision. CONCLUSIONS: In this study, CP-675,206 immunotherapy for metastatic melanoma did not adversely affect the eye or vision.

The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.