Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3'-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids.

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Potential of several triazene derivatives against DENGUE viruses.

Dengue fever is an infectious disease caused by the dengue virus (DENV), an RNA Flavivirus transmitted by the mosquitoes Aedes aegypti and Aedes albopictus widespread in tropical, subtropical and also temperate regions. Symptoms range from a simple cold to a severe, life-threatening haemorrhagic fever. According to the WHO, it affects around 390 million people per year. No antiviral treatment for DENV is available, and the Dengvaxia vaccine is only intended for people over 9 years of age who have contracted dengue one time in the past, and shows serotype-specific effectiveness. There is therefore a crying need to discover new molecules with antiviral power against flaviviruses. The present study was carried out to evaluate the anti-DENV activities and cytotoxicity of triazenes obtained by diazocopulation. Some triazenes were highly cytotoxic (16, and 25) to hepatocarcinoma Huh7 cells, whereas others displayed strong anti-DENV potential. The antiviral activity ranged from EC50 = 7.82 µM to 48.12 µM in cellulo, with a selectivity index (CC50/EC50) greater than 9 for two of the compounds (10, and 20). In conclusion, these new triazenes could serve as a lead to develop and optimize drugs against DENV.

Biochemical Analyses of Bioactive Extracts from Plants Native to Lampedusa, Sicily Minor Island.

Major threats to the human lifespan include cancer, infectious diseases, diabetes, mental degenerative conditions and also reduced agricultural productivity due to climate changes, together with new and more devastating plant diseases. From all of this, the need arises to find new biopesticides and new medicines. Plants and microorganisms are the most important sources for isolating new metabolites. Lampedusa Island host a rich contingent of endemic species and subspecies. Seven plant species spontaneously growing in Lampedusa, i.e., Atriplex halimus L. (Ap), Daucus lopadusanus Tineo (Dl), Echinops spinosus Fiori (Es) Glaucium flavum Crantz (Gf) Hypericum aegypticum L: (Ha), Periploca angustifolia Labill (Pa), and Prasium majus L. (Pm) were collected, assessed for their metabolite content, and evaluated for potential applications in agriculture and medicine. The HPLC-MS analysis of n-hexane (HE) and CH2Cl2 (MC) extracts and the residual aqueous phases (WR) showed the presence of several metabolites in both organic extracts. Crude HE and MC extracts from Dl and He significantly inhibited butyrylcholinesterase, as did WR from the extraction of Dl and Pa. HE and MC extracts showed a significant toxicity towards hepatocarcinoma Huh7, while Dl, Ha and Er HE extracts were the most potently cytotoxic to ileocecal colorectal adenocarcinoma HCT-8 cell lines. Most extracts showed antiviral activity. At the lowest concentration tested (1.56 µg/mL), Dl, Gf and Ap MC extracts inhibited betacoronavirus HCoV-OC43 infection by> 2 fold, while the n-hexane extract of Pm was the most potent. In addition, at 1.56 µg/mL, potent inhibition (>10 fold) of dengue virus was detected for Dl, Er, and Pm HE extracts, while Pa and Ap MC extracts dampened infections to undetectable levels. Regarding to phytotoxicity, MC extracts from Er, Ap and Pm were more effective in inhibiting tomato rootlet elongation; the same first two extracts also inhibited seed cress germination while its radicle elongation, due to high sensitivity, was affected by all the extracts. Es and Gf MC extracts also inhibited seed germination of Phelipanche ramosa. Thus, we have uncovered that many of these Lampedusa plants displayed promising biopesticide, antiviral, and biological properties.

Chemical Synthesis and Biological Activities of Amaryllidaceae Alkaloid Norbelladine Derivatives and Precursors.

Amaryllidaceae alkaloids (AAs) are a structurally diverse family of alkaloids recognized for their many therapeutic properties, such as antiviral, anti-cholinesterase, and anticancer properties. Norbelladine and its derivatives, whose biological properties are poorly studied, are key intermediates required for the biosynthesis of all ~650 reported AAs. To gain insight into their therapeutic potential, we synthesized a series of O-methylated norbelladine-type alkaloids and evaluated their cytotoxic effects on two types of cancer cell lines, their antiviral effects against the dengue virus (DENV) and the human immunodeficiency virus 1 (HIV-1), and their anti-Alzheimer's disease (anti-cholinesterase and -prolyl oligopeptidase) properties. In monocytic leukemia cells, norcraugsodine was highly cytotoxic (CC50 = 27.0 µM), while norbelladine was the most cytotoxic to hepatocarcinoma cells (CC50 = 72.6 µM). HIV-1 infection was impaired only at cytotoxic concentrations of the compounds. The 3,4-dihydroxybenzaldehyde (selectivity index (SI) = 7.2), 3',4'-O-dimethylnorbelladine (SI = 4.8), 4'-O-methylnorbelladine (SI > 4.9), 3'-O-methylnorbelladine (SI > 4.5), and norcraugsodine (SI = 3.2) reduced the number of DENV-infected cells with EC50 values ranging from 24.1 to 44.9 µM. The O-methylation of norcraugsodine abolished its anti-DENV potential. Norbelladine and its O-methylated forms also displayed butyrylcholinesterase-inhibition properties (IC50 values ranging from 26.1 to 91.6 µM). Altogether, the results provided hints of the structure−activity relationship of norbelladine-type alkaloids, which is important knowledge for the development of new inhibitors of DENV and butyrylcholinesterase.

Cytotoxicity and Antiviral Properties of Alkaloids Isolated from Pancratium maritimum.

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identified as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O'-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Lycorine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 µM and 18.5 µM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC50 ranged from 0.34−73.59 µM) at low to non-cytotoxic concentrations (CC50 ranged from 6.25 µM to >100 µM). Haemanthamine (EC50 = 337 nM), pancracine (EC50 = 357 nM) and haemanthidine (EC50 = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases.

Biological Investigation of Amaryllidaceae Alkaloid Extracts from the Bulbs of Pancratium trianthum Collected in the Senegalese Flora.

Amaryllidaceae plants are rich in alkaloids with biological properties. Pancratium trianthum is an Amaryllidaceae species widely used in African folk medicine to treat several diseases such as central nervous system disorders, tumors, and microbial infections, and it is used to heal wounds. The current investigation explored the biological properties of alkaloid extracts from bulbs of P. trianthum collected in the Senegalese flora. Alkaloid extracts were analyzed and identified by chromatography and mass spectrometry. Alkaloid extracts from P. trianthum displayed pleiotropic biological properties. Cytotoxic activity of the extracts was determined on hepatocarcinoma Huh7 cells and on acute monocytic leukemia THP-1 cells, while agar diffusion and microdilution assays were used to evaluate antibacterial activity. Antiviral activity was measured by infection of extract-treated cells with dengue virus (DENVGFP) and human immunodeficiency virus-1 (HIV-1GFP) reporter vectors. Cytotoxicity and viral inhibition were the most striking of P. trianthum's extract activities. Importantly, non-cytotoxic concentrations were highly effective in completely preventing DENVGFP replication and in reducing pseudotyped HIV-1GFP infection levels. Our results show that P. trianthum is a rich source of molecules for the potential discovery of new treatments against various diseases. Herein, we provide scientific evidence to rationalize the traditional uses of P. trianthum for wound treatment as an anti-dermatosis and antiseptic agent.

Isolation and Biological Characterization of Homoisoflavanoids and the Alkylamide N-p-Coumaroyltyramine from Crinum biflorum Rottb., an Amaryllidaceae Species Collected in Senegal.

Crinum biflorum Rottb. (syn. Crinum distichum) is an Amaryllidaceae plant used in African traditional medicine but very few studies have been performed on this species from a chemical and applicative point of view. Bulbs of C. biflorum, collected in Senegal, were extracted with ethanol by Soxhlet and the corresponding organic extract was purified using chromatographic methods. The pure compounds were chemically characterized by spectroscopic techniques (1D and 2D 1H and 13C NMR, HR MS and ECD) and X-ray analysis. Four homoisoflavonoids (1-4) and one alkylamide (5) were isolated and characterized as 5,6,7-trimethoxy-3-(4-hydroxybenzyl)chroman-4-one (1), as 3-hydroxy-5,6,7-trimethoxy-3-(4-hydroxybenzyl)chroman-4-one (2), as 3-hydroxy-5,6,7-trimethoxy-3-(4-methoxybenzyl)chroman-4-one (3) and as 5,6,7-trimethoxy-3-(4-methoxybenzyl)chroman-4-one (4), and the alkylamide as (E)-N-(4-hydroxyphenethyl)-3-(4-hydroxyphenyl)acrylamide (5), commonly named N-p-coumaroyltyramine. The relative configuration of compound 1 was verified thanks to the X-ray analysis which also allowed us to confirm its racemic nature. The absolute configurations of compounds 2 and 3 were assigned by comparing their ECD spectra with those previously reported for urgineanins A and B. Flavanoids 1, 3 and 4 showed promising anticancer properties being cytotoxic at low micromolar concentrations towards HeLa and A431 human cancer cell lines. The N-p-coumaroyltyramine (5) was selectively toxic to A431 and HeLa cancer cells while it protected immortalized HaCaT cells against oxidative stress induced by hydrogen peroxide. Compounds 1-4 also inhibited acetylcholinesterase activity with compound 3 being the most potent. The anti-amylase and the strong anti-glucosidase activity of compound 5 were confirmed. Our results show that C. biflorum produces compounds of therapeutic interest with anti-diabetic, anti-tumoral and anti-acetylcholinesterase properties.

Biosynthesis and Biological Activities of Newly Discovered Amaryllidaceae Alkaloids.

Alkaloids are an important group of specialized nitrogen metabolites with a wide range of biochemical and pharmacological effects. Since the first publication on lycorine in 1877, more than 650 alkaloids have been extracted from Amaryllidaceae bulbous plants and clustered together as the Amaryllidaceae alkaloids (AAs) family. AAs are specifically remarkable for their diverse pharmaceutical properties, as exemplified by the success of galantamine used to treat the symptoms of Alzheimer's disease. This review addresses the isolation, biological, and structure activity of AAs discovered from January 2015 to August 2020, supporting their therapeutic interest.

A First Insight into North American Plant Pathogenic Fungi Armillaria Sinapina Transcriptome.

Armillaria sinapina, a fungal pathogen of primary timber species of North American forests, causes white root rot disease that ultimately kills the trees. A more detailed understanding of the molecular mechanisms underlying this illness will support future developments on disease resistance and management, as well as in the decomposition of cellulosic material for further use. In this study, RNA-Seq technology was used to compare the transcriptome profiles of A. sinapina fungal culture grown in yeast malt broth medium supplemented or not with betulin, a natural compound of the terpenoid group found in abundance in white birch bark. This was done to identify enzyme transcripts involved in the metabolism (redox reaction) of betulin into betulinic acid, a potent anticancer drug. De novo assembly and characterization of A. sinapina transcriptome was performed using Illumina technology. A total of 170,592,464 reads were generated, then 273,561 transcripts were characterized. Approximately, 53% of transcripts could be identified using public databases with several metabolic pathways represented. A total of 11 transcripts involved in terpenoid biosynthesis were identified. In addition, 25 gene transcripts that could play a significant role in lignin degradation were uncovered, as well as several redox enzymes of the cytochromes P450 family. To our knowledge, this research is the first transcriptomic study carried out on A. sinapina.

[Cellular and antiviral functions of TRIM5α]. / Fonctions cellulaires et antivirales de TRIM5α.

In this review, we summarize recent advances in the knowledge of the biological functions of human TRIM5α, a cytoplasmic protein mostly known for its antiretroviral functions. In addition to directly targeting retroviral capsid cores, an inhibitory activity called "restriction", TRIM5α senses retroviruses and activates NF-κB and AP-1 signaling pathways, resulting in the production of type I interferon (IFN-I). The antiviral state resulting from the activation of these pathways includes the upregulation of other restriction factors, and is thought to be important for the control of HIV-1 in some patients. TRIM5α also targets the protease enzyme of several tick-borne flaviviruses, a family of viruses not closely related to retroviruses. In addition to these antiviral functions, TRIM5α promotes autophagy by interacting with key actors of this pathway, such as ULK1 and p62. TRIM5α may function as a selective autophagy receptor in some conditions. Altogether, our understanding of TRIM5α shows its potential for the development of medical applications in viral diseases and beyond.

Gene Knockout Shows That PML (TRIM19) Does Not Restrict the Early Stages of HIV-1 Infection in Human Cell Lines.

The PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. In contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNA interference, with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used the clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9) system to knock out its gene in epithelial, lymphoid, and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Overexpression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5α (another human TRIM protein), and TRIM5α was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages. IMPORTANCE PML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide unambiguous, knockout-based evidence that PML does not restrict the early postentry stages of HIV-1 infection in a variety of human cell types and does not participate in the inhibition of HIV-1 by IFN-I. Although this study does not exclude the possibility of other mechanisms by which PML may interfere with HIV-1, we nonetheless demonstrate that PML does not generally act as an HIV-1 restriction factor in human cells and that its presence is not required for IFN-I to stimulate the expression of anti-HIV-1 genes. These results contribute to uncovering the landscape of HIV-1 inhibition by ISGs in human cells.

The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

BACKGROUND: The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. RESULTS: We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-ß treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. CONCLUSIONS: We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has implications for the development of murine models of HIV-1.

Development of an IFN-γ ELISpot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation.

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-γ production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens.  

Reconstitution of protective immune responses against cytomegalovirus and varicella zoster virus does not require disease development in pediatric recipients of umbilical cord blood transplantation.

CMV and varicella zoster virus (VZV) are significant causes of morbidity and mortality following umbilical cord blood transplantation (UCBT). However, the kinetics of reconstitution and protective potential of antiviral cell-mediated immune responses following UCBT remain poorly characterized. In this study, the reconstitution of CMV- and VZV-specific T cell responses was assessed using IFN-γ ELISPOT in 28 children who underwent UCBT to treat hematological or inherited disorders. Barely detectable in the first 3 mo posttransplantation, CMV- and VZV-specific T cell responses were observed in 30.4% and 40.3% of study subjects after 36 mo of follow-up. Four of five CMV-seropositive subjects developed detectable levels of circulating CMV DNA (DNAemia), and 5 of 17 VZV-seropositive patients experienced herpes zoster during the posttransplant period. Four CMV-seronegative subjects developed IFN-γ responses against CMV, and four subjects developed a VZV-specific IFN-γ response without clinical signs of infection. No CMV- or VZV-related events were observed in study subjects following the development of CMV- or VZV-specific responses > 150 spot-forming units/10(6) PBMCs, consistent with T cell-mediated protection. Finally, famciclovir prophylaxis did not strictly prevent the reconstitution of the VZV-specific T cell repertoire, because the frequency of T cells producing IFN-γ in response to VZV Ags reached levels consistent with protection in two nonzoster subjects. Monitoring of CMV- and VZV-specific cell-mediated immunity could inform immunocompetence and guide the initiation and cessation of antiherpetic prophylaxis in UCBT recipients.

CD8(+) T-cell reconstitution in recipients of umbilical cord blood transplantation and characteristics associated with leukemic relapse.

Recipients of umbilical cord blood (UCB) transplantation (UCBT) face a high risk of morbidity and mortality related to opportunistic infections (OI) and leukemic relapse. To understand the molecular basis of these UCBT-related complications, the characteristics of UCB-derived antigen-specific CD8(+) T cells were examined in a group of pediatric UCBT recipients. Compared with the UCB graft inoculum and the late post-UCBT period (12-36 months), declining clonal diversity of UCB-derived CD8(+) T cells specific for the Melan-A(26-35) A27L peptide and high frequencies of PD-1-expressing CD8(+) T cells were observed in the first 3 months after UCBT, a period during which OIs are most frequent. The CD8(+) T-cell compartment predominantly comprised CD45RA(+) CCR7(-) terminally differentiated effector-memory T cells until 6 months after UCBT, at which time the polyfunctionality of antigen-specific CD8(+) T cells was reestablished. Finally, the frequency of PD-1(+) CD8(+) T cells was significantly higher in subjects who subsequently experienced leukemic relapse. This study informs the biologic properties of UCB-derived CD8(+) T cells and provides a rationale for the characteristics of UCBT in terms of immune reconstitution and OI. These results also suggest that the elevated frequency of PD-1(+) CD8(+) T cells could be associated with leukemic relapse in pediatric UCBT recipients.

Complementary and contrasting roles of NK cells and T cells in pediatric umbilical cord blood transplantation.

UCBT has been used for almost 25 years to treat a variety of malignant and nonmalignant childhood diseases. The biological properties of NK cells and T cells and their implication in engraftment, immune reconstitution, OIs, leukemic relapse, and GvHD have been explored in the context of UCBT. These studies have established that lymphocytes have a major impact on the outcome of UCBT and that NK cells and T cells play complementary and contrasting roles in immune reconstitution and the GvL effect. Therefore, novel strategies to improve the outcome of UCBT recipients, including immunotherapeutic regimens, should be based on key immunologic features of UCB T lymphocytes and NK cells.

Umbilical cord blood T cells respond against the Melan-A/MART-1 tumor antigen and exhibit reduced alloreactivity as compared with adult blood-derived T cells.

Umbilical cord blood (UCB) is increasingly used as a source of hematopoietic progenitor cells to treat a variety of disorders. UCB transplant is associated with comparatively reduced incidence of graft-versus-host disease, robust graft versus leukemia effect, and relatively high incidence of opportunistic infections, three processes in which donor-derived T lymphocytes are known to be predominantly involved. To examine the differential functionality of UCB T cells, CD8(+) T cells specific for the melanoma-associated HLA-A2-restricted Melan-A(26-35) A27L peptide were isolated from HLA-A2(+) and HLA-A2(-) UCB samples and HLA-A2(+) and HLA-A2(-) adult peripheral blood using A2/Melan-A tetramers. In UCB samples, A2/Melan-A(+) CD8(+) T cells were detected at a frequency of 0.04%, were more frequent in HLA-A2(+) UCB, and were polyclonal and mostly naive. Consistent with Ag-driven expansion, the frequency of A2/Melan-A(+) CD8(+) T cells was increased following stimulation with cognate peptide or polyclonal activation, they acquired cell-surface markers reflective of effector/memory differentiation, their TCR repertoire became oligoclonal, and they expressed cytolytic activity and produced IFN-gamma. Although functional properties of A2/Melan-A(+) CD8(+) T cells derived from HLA-A2(+) UCB resembled those of HLA-A2(+) adult peripheral blood, they were more likely to reach terminal differentiation following polyclonal stimulation and produced less IFN-gamma in response to cognate peptide. A2/Melan-A(+) CD8(+) T cells from HLA-A2(-) UCB were poorly cytolytic, produced little IFN-gamma, and were predominantly monofunctional or nonfunctional. These properties of UCB-derived CD8(+) T cells could contribute to the reduced incidence of graft-versus-host disease and heightened incidence of opportunistic infections observed following UCB transplant.

HIV-1 genetic diversity in antenatal cohort, Canada.

We studied HIV genetic diversity in a cohort of 127 pregnant, HIV-infected women who received prenatal care at Sainte-Justine Hospital in Montreal, Canada, between 1999 and 2003. Clade assignments were derived by phylogenetic analysis of amplified pol sequences. Genotyping was successful in 103 of 127 women, 59 (57.3%) of whom were infected with clade B HIV-1, and 44 (42.7%) with nonclade B viruses, including subtypes A, C, D, F, G, and H. Four sequences remained unassigned. Forty-three of 44 women infected with non-clade B viruses were newcomers from sub-Saharan Africa, and subtype identity was consistent with those circulating in their countries of origin. These results highlight the epidemiologic importance of non-B HIV-1 in antenatal populations in a large North American urban center, underscore the influence of population movements on clade intermixing, and identify a group of patients who could be targeted for surveillance and drug therapy followup.