Telomouse-a mouse model with human-length telomeres generated by a single amino acid change in RTEL1.

Telomeres, the ends of eukaryotic chromosomes, protect genome integrity and enable cell proliferation. Maintaining optimal telomere length in the germline and throughout life limits the risk of cancer and enables healthy aging. Telomeres in the house mouse, Mus musculus, are about five times longer than human telomeres, limiting the use of this common laboratory animal for studying the contribution of telomere biology to aging and cancer. We identified a key amino acid variation in the helicase RTEL1, naturally occurring in the short-telomere mouse species M. spretus. Introducing this variation into M. musculus is sufficient to reduce the telomere length set point in the germline and generate mice with human-length telomeres. While these mice are fertile and appear healthy, the regenerative capacity of their colonic epithelium is compromised. The engineered Telomouse reported here demonstrates a dominant role of RTEL1 in telomere length regulation and provides a unique model for aging and cancer.

High Prevalence of Clonal Reproduction and Low Genetic Diversity in Scutellaria floridana, a Federally Threatened Florida-Endemic Mint.

The threatened mint Florida skullcap (Scutellaria floridana) is endemic to four counties in the Florida panhandle. Because development and habitat modification extirpated several historical occurrences, only 19 remain to date. To inform conservation management and delisting decisions, a comprehensive investigation of the genetic diversity and relatedness, population structure, and clonal diversity was conducted using SNP data generated by ddRAD. Compared with other Lamiaceae, we detected low genetic diversity (HE = 0.125-0.145), low to moderate evidence of inbreeding (FIS = -0.02-0.555), and moderate divergence (FST = 0.05-0.15). We identified eight populations with most of the genetic diversity, which should be protected in situ, and four populations with low genetic diversity and high clonality. Clonal reproduction in our circular plots and in 92% of the sites examined was substantial, with average clonal richness of 0.07 and 0.59, respectively. Scutellaria floridana appears to have experienced a continued decline in the number of extant populations since its listing under the Endangered Species Act; still, the combination of sexual and asexual reproduction may be advantageous for maintaining the viability of extant populations. However, the species will likely require ongoing monitoring, management, and increased public awareness to ensure its survival and effectively conserve its genetic diversity.

Highly Multiplexed Image Analysis of Intestinal Tissue Sections in Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Significant progress has been made since the first report of inflammatory bowel disease (IBD) in 1859, after decades of research that have contributed to the understanding of the genetic and environmental factors involved in IBD pathogenesis. Today, a range of treatments is available for directed therapy, mostly targeting the overactive immune response. However, the mechanisms by which the immune system contributes to disease pathogenesis and progression are not fully understood. One challenge hindering IBD research is the heterogeneous nature of the disease and the lack of understanding of how immune cells interact with one another in the gut mucosa. Introduction of a technology that enables expansive characterization of the inflammatory environment of human IBD tissues may address this gap in knowledge. METHODS: We used the imaging mass cytometry platform to perform highly multiplex image analysis of IBD and healthy deidentified intestine sections (6 Crohn's disease compared to 6 control ileum; 6 ulcerative colitis compared to 6 control colon). The acquired images were graded for inflammation severity by analysis of adjacent H&E tissue sections. We assigned more than 300,000 cells to unique cell types and performed analyses of tissue integrity, epithelial activity, and immune cell composition. RESULTS: The intestinal epithelia of patients with IBD exhibited increased proliferation rates and expression of HLA-DR compared to control tissues, and both features were positively correlated with the severity of inflammation. The neighborhood analysis determined enrichment of regulatory T cell interactions with CD68+ macrophages, CD4+ T cells, and plasma cells in both forms of IBD, whereas activated lysozyme C+ macrophages were preferred regulatory T cell neighbors in Crohn's disease but not ulcerative colitis. CONCLUSIONS: Altogether, our study shows the power of imaging mass cytometry and its ability to both quantify immune cell types and characterize their spatial interactions within the inflammatory environment by a single analysis platform.

Blinding in Clinical Trials for Chronic Liver Diseases.

Within the field of randomized clinical trials (RCTs), the randomized double-blind placebo-controlled clinical trial is considered the most efficient means of simultaneously assessing the efficacy and safety of a medical therapy in a single trial. While many RCTs are conducted without blinding (open label), it is rare to encounter a blinded trial that does not randomize its subjects. Clinical trials for chronic liver diseases have adopted many of the practices set forth by RCTs in other chronic diseases, but blinding has often been difficult to properly implement. This review examines the rationale for blinding, common challenges to successful blinding, different mechanisms of unintentional unblinding in clinical trials for viral hepatitis and nonalcoholic steatohepatitis, and recommendations for blinding and design in future trials of treatments for liver disease.

Pleistocene aridification underlies the evolutionary history of the Caribbean endemic, insular, giant Consolea (Opuntioideae).

PREMISE: The Caribbean islands are in the top five biodiversity hotspots on the planet; however, the biogeographic history of the seasonally dry tropical forest (SDTF) there is poorly studied. Consolea consists of nine species of dioecious, hummingbird-pollinated tree cacti endemic to the West Indies, which form a conspicuous element of the SDTF. Several species are threatened by anthropogenic disturbance, disease, sea-level rise, and invasive species and are of conservation concern. However, no comprehensive phylogeny yet exists for the clade. METHODS: We reconstructed the phylogeny of Consolea, sampling all species using plastomic data to determine relationships, understand the evolution of key morphological characters, and test their biogeographic history. We estimated divergence times to determine the role climate change may have played in shaping the current diversity of the clade. RESULTS: Consolea appears to have evolved very recently during the latter part of the Pleistocene on Cuba/Hispaniola likely from a South American ancestor and, from there, moved into the Bahamas, Jamaica, Puerto Rico, Florida, and the Lesser Antilles. The tree growth form is a synapomorphy of Consolea and likely aided in the establishment and diversification of the clade. CONCLUSIONS: Pleistocene aridification associated with glaciation likely played a role in shaping the current diversity of Consolea, and insular gigantism may have been a key innovation leading to the success of these species to invade the often-dense SDTF. This in-situ Caribbean radiation provides a window into the generation of species diversity and the complexity of the SDTF community within the Antilles.

Bioactive Plasma Mitochondrial DNA Is Associated With Disease Progression in Scleroderma-Associated Interstitial Lung Disease.

OBJECTIVE: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is characterized by variable clinical outcomes, activation of innate immune pattern-recognition receptors (PRRs), and accumulation of α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs Toll-like receptor 9 (TLR-9) and cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. METHODS: Human lung fibroblasts (HLFs) from normal donors and SSc-ILD explants were treated with synthetic CpG DNA and assayed for α-SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in 2 independent SSc-ILD cohorts and demographically matched controls. The ability of SSc-ILD and control plasma to induce TLR-9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin-6 (IL-6), and oxidized DNA were measured using electrochemiluminescence and enzyme-linked immunosorbent assay-based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. RESULTS: Normal HLFs and SSc-ILD fibroblasts developed increased α-SMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc-ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR-9 and cGAS/STING activation as well as type I IFN and IL-6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. CONCLUSION: These findings demonstrate a previously unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

Helicobacter pylori antimicrobial resistance and antibiotic consumption in the low-resource Central America setting.

BACKGROUND: Antimicrobial resistance is a global public health problem, particularly in low- and middle-income countries (LMICs), where antibiotics are often obtained without a prescription. H. pylori antimicrobial resistance patterns are informative for patient care and gastric cancer prevention programs, have been shown to correlate with general antimicrobial consumption, and may guide antimicrobial stewardship programs in LMICs. We report H. pylori resistance and antimicrobial utilization patterns for western Honduras, representative of rural Central America. METHODS: In the context of the western Honduras gastric cancer epidemiology initiative, gastric biopsies from 189 patients were studied for culture and resistance patterns. Antimicrobial utilization was investigated for common H. pylori treatment regimens from regional public (7 antimicrobials) and national private (4 antimicrobials) data, analyzed in accordance with WHO anatomical therapeutic chemical defined daily doses (DDD) method and expressed as DDD/1000 inhabitants per day (DID) and per year (DIY). RESULTS: H. pylori was successfully cultured from 116 patients (56% males, mean age: 54), and nearly all strains were cagA+ and vacAs1m1+ positive (99% and 90.4%, respectively). Unexpectedly, high resistance was noted for levofloxacin (20.9%) and amoxicillin (10.7%), while metronidazole (67.9%) and clarithromycin (11.2%) were similar to data from Latin America. Significant associations with age, gender, or histology were not noted, with the exception of levofloxacin (28%, P = 0.01) in those with histology limited to non-atrophic gastritis. Total antimicrobial usage in western Honduras of amoxicillin (17.3 DID) and the quinolones had the highest relative utilizations compared with other representative nations. CONCLUSIONS: We observed significant H. pylori resistance to amoxicillin and levofloxacin in the context of high community antimicrobial utilization. This has implications in Central America for H. pylori treatment guidelines as well as antimicrobial stewardship programs.

Antropometría nutricional en niños de uno a seis años malnutridos por exceso / Nutritional anthropometry in children from one to six year old mal nourished by excess

Fundamento: la obesidad es uno de los trastornos crónicos más comunes de la niñez, con una prevalencia que aumenta de manera rápida y continua.Objetivo: describir algunas características antropométricas en niños de uno a seis años con malnutrición por exceso.Métodos: se realizó un estudio observacional descriptivo y transversal en el departamento de Anatomía de Ciencias Médicas de Camagüey, desde marzo de 2015 hasta mayo de 2016. El universo estuvo conformado por 482 niños pertenecientes a los Círculos Infantiles del municipio, la muestra quedó conformada por 101 malnutridos por exceso. Las variables estudiadas fueron: edad, sexo, peso, talla, índice de masa corporal, perímetro braquial, pliegues cutáneos tricipital, subescapular y suprailíaco. El análisis estadístico se realizó mediante el paquete estadístico SPSS. Se aplicó la antropometría como técnica y se utilizaron las tablas de evaluación nutricional vigentes para la Atención Primaria de Salud.Resultados: predominó la categoría de obeso y sobrepeso con una incidencia notable para todos los grupos de edades, donde la mayor incidencia fue para el sexo masculino con los mayores porcientos.Conclusiones: se encontraron cifras significativas del peso e índice de masa corporal con mayor incidencia en los varones obesos que en las hembras, para estas últimas la frecuencia fue mayor en la sumatoria de los pliegues cutáneos. Existió diferencia significativa en las medias del peso y la talla entre los tres grupos de edades, mientras que el perímetro braquial fue diferente entre los grupos de edades extremos y entre el de uno a dos años con el de tres a cuatro años(AU)

Background: obesity is one of the commonest chronic disorders in childhood, with a prevalence that increases rapidly and continuously.Objective: to describe some of the anthropometric features in children from one to six year old who present malnourishment by excess.Methods: an observational, descriptive and transversal study was conducted in the Human Anatomy Department at the School of Medicine in Camaguey from March 2015 to May 2016. The universe was composed of 482 children who belong to the nurseries in Vertientes municipality. The sample was 101 children who are malnourished by excess. The variables studied were: age, weight, height, BMI, brachial perimeter, tricipital, bicipital, subscapular and suprailiac skinfolds. Statistical analysis was carried out using the SPSS program. Anthropometric technique was performed and nutritional tables were used by physicians at primary health care nowadays to evaluate the nutritional condition.Results: predominance of obesity and overweight with a remarkable incidence for all groups of age, where the greater incidence was for males with the biggest percentages.Conclusions: significant figures in weight and BMI were found, with more impact in obese male than in females. For the last ones the frequency was greater in all the skin folds. There were significant differences in terms of weight and height between the three groups of age, meanwhile the brachial perimeter was different between extreme groups of age and between the one to two year age group with three to four year age group(AU)

Antropometría nutricional en niños de uno a seis años malnutridos por exceso / Nutritional anthropometry in children from one to six year old mal nourished by excess

Fundamento: la obesidad es uno de los trastornos crónicos más comunes de la niñez, con una prevalencia que aumenta de manera rápida y continua. Objetivo: describir algunas características antropométricas en niños de uno a seis años con malnutrición por exceso. Métodos: se realizó un estudio observacional descriptivo y transversal en el departamento de Anatomía de Ciencias Médicas de Camagüey, desde marzo de 2015 hasta mayo de 2016. El universo estuvo conformado por 482 niños pertenecientes a los Círculos Infantiles del municipio, la muestra quedó conformada por 101 malnutridos por exceso. Las variables estudiadas fueron: edad, sexo, peso, talla, índice de masa corporal, perímetro braquial, pliegues cutáneos tricipital, subescapular y suprailíaco. El análisis estadístico se realizó mediante el paquete estadístico SPSS. Se aplicó la antropometría como técnica y se utilizaron las tablas de evaluación nutricional vigentes para la Atención Primaria de Salud. Resultados: predominó la categoría de obeso y sobrepeso con una incidencia notable para todos los grupos de edades, donde la mayor incidencia fue para el sexo masculino con los mayores porcientos. Conclusiones: se encontraron cifras significativas del peso e índice de masa corporal con mayor incidencia en los varones obesos que en las hembras, para estas últimas la frecuencia fue mayor en la sumatoria de los pliegues cutáneos. Existió diferencia significativa en las medias del peso y la talla entre los tres grupos de edades, mientras que el perímetro braquial fue diferente entre los grupos de edades extremos y entre el de uno a dos años con el de tres a cuatro años.

Background: obesity is one of the commonest chronic disorders in childhood, with a prevalence that increases rapidly and continuously. Objective: to describe some of the anthropometric features in children from one to six year old who present malnourishment by excess. Methods: an observational, descriptive and transversal study was conducted in the Human Anatomy Department at the School of Medicine in Camaguey from March 2015 to May 2016. The universe was composed of 482 children who belong to the nurseries in Vertientes municipality. The sample was 101 children who are malnourished by excess. The variables studied were: age, weight, height, BMI, brachial perimeter, tricipital, bicipital, subscapular and suprailiac skinfolds. Statistical analysis was carried out using the SPSS program. Anthropometric technique was performed and nutritional tables were used by physicians at primary health care nowadays to evaluate the nutritional condition. Results: predominance of obesity and overweight with a remarkable incidence for all groups of age, where the greater incidence was for males with the biggest percentages. Conclusions: significant figures in weight and BMI were found, with more impact in obese male than in females. For the last ones the frequency was greater in all the skin folds. There were significant differences in terms of weight and height between the three groups of age, meanwhile the brachial perimeter was different between extreme groups of age and between the one to two year age group with three to four year age group.

Chronic ethanol diet increases regulatory T-cell activity and inhibits hepatitis C virus core-specific cellular immune responses in mice.

AIM: Chronic ethanol consumption is associated with persistent hepatitis C viral (HCV) infection. This study explores the role of the host cellular immune response to HCV core protein in a murine model and how chronic ethanol consumption alters T-cell regulatory (Treg) populations. METHODS: BALB/c mice were fed an isocaloric control or ethanol liquid diet. Dendritic cells (DC) were isolated after expansion with a hFl3tL-expression plasmid and subsequently transfected with HCV core protein. Core-containing DC (1 × 10(6) ) were s.c. injected (×3) in mice every 2 weeks. Splenocytes from immunized mice were isolated and stimulated with HCV core protein to measure generation of viral antigen-specific Treg, as well as secretion of interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-4. Cytotoxicity was measured by lactate dehydrogenase release from HCV core-expressing syngeneic SP2/19 myeloma cells. RESULTS: Splenocytes from mice immunized with ethanol-derived and HCV core-loaded DC exhibited significantly lower in vitro cytotoxicity compared to mice immunized with HCV core-loaded DC derived from isocaloric pair-fed controls. Stimulation with HCV core protein triggered higher IL-2, TNF-α and IL-4 release in splenocytes following immunization with core-loaded DC derived from controls as compared to chronic ethanol-fed mice. Splenocytes derived from mice immunized with core-loaded DC isolated from ethanol-fed mice exhibited a significantly higher CD25(+) FOXP3(+) and CD4(+) FOXP3(+) Treg population. CONCLUSION: These results suggest that immunization with HCV core-containing DC from ethanol-fed mice induces an increase in the CD25(+) FOXP3(+) and CD4(+) FOXP3(+) Treg population and may suppress HCV core-specific CD4(+) and CD8(+) T-cell immune responses.

Immunization with aspartate-ß-hydroxylase-loaded dendritic cells produces antitumor effects in a rat model of intrahepatic cholangiocarcinoma.

UNLABELLED: Dendritic cells (DCs) capture and process proteins and present peptides on the cell surface in the context of major histocompatibility complex I and II molecules to induce antigen-specific T cell immune responses. The aims of this study were to (1) employ an expanded and purified DC population and load them with aspartate-ß-hydroxylase (ASPH), a highly expressed tumor-associated cell surface protein, and (2) to determine if immunization induced antitumor effects in an orthotopic rat model of intrahepatic cholangiocarcinoma. Splenocytes were incubated with ASPH-coated beads and passed through a magnetic field to yield an 80% pure DC OX62+ population. This DC subset was stimulated with granulocyte-macrophage colony-stimulating factor, interleukin-4, CD40L, and interferon-γ, resulting in a 40-fold increase in interleukin-12A messenger RNA expression to subsequently generate a T helper 1-type immune response. After incubation with the cytokine cocktail, DCs were found to have matured, as demonstrated by increased expression of CD40, CD80, and CD86 costimulatory molecules. Immunization with ASPH-loaded DCs induced antigen-specific immunity. A clone of the parental tumorigenic rat BDEneu cholangiocyte cell line, designated BDEneu-CL24, was found to have the highest number of cells expressing this surface protein (97%); it maintained the same phenotypic characteristics of the parental cell line and was used to produce intrahepatic tumors in immunocompetent syngeneic Fisher-344 rats. Immunization with ASPH-loaded DCs generated cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed intrahepatic tumor growth and metastasis, and was associated with increased CD3+ lymphocyte infiltration into the tumors. CONCLUSION: These findings suggest that immunization with ASPH-loaded DCs may constitute a novel therapeutic approach for intrahepatic cholangiocarcinoma, because this protein also appears to be highly conserved and expressed on human hepatobiliary tumors.

Chronic alcohol-induced liver disease inhibits dendritic cell function.

BACKGROUND/AIMS: We have compared dendritic cell (DC) function derived from the alcoholic liver disease (ALD) sensitive Long-Evans (LE) and resistant Fischer rat strains to determine if the influence of ethanol on DCs was dependent on ALD. METHODS: The LE and Fischer rats were fed an ethanol-containing or isocaloric control liquid diet for 8 weeks and comparisons were made to LE rats injected with thioacetamide as a liver disease control. DCs were isolated from the spleen after expansion with human Fms-like tyrosine kinase receptor 3 ligand plasmid. Maturation markers CD86, CD80, CD40 and MHC-II were analysed by flow cytometry with or without lipopolysaccharide and poly I:C stimulation. Production of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-12p40 and IL-10 cytokines and the antigen presentation ability of DCs was determined. RESULTS: Only LE rats developed ALD characterized by liver injury, elevated alanine aminotransferase levels and steatosis; CD86 and CD40 expression was decreased in LE but not Fischer rats. Reduced TNF-α, IFN-γ, IL-12, proinflammatory and enhanced IL-10 cytokine production was found in DCs isolated from ethanol-fed LE but not Fischer rats. Allostimulatory activity was reduced in LE compared with the Fischer strain. In contrast, DCs isolated from thioacetamide-induced liver damage displayed a reduction only in IL-12p40; TNF-α, IL-10 and IFN-α production as well as antigen presenting ability remained intact compared with controls. CONCLUSIONS: ALD sensitive LE rats exhibited characteristics of a suppressed DC phenotype that was not observed following thioacetamide-induced liver disease, which suggests an important role for ALD in altering the host cellular and humoral immune responses.

Nanomaterials for X-ray imaging: gold nanoparticle enhancement of X-ray scatter imaging of hepatocellular carcinoma.

We present the development of a new imaging technique for the early diagnosis of hepatocellular carcinoma that utilizes surface-modified gold nanoparticles in combination with X-ray imaging. Tissues labeled with these electron-dense particles show enhanced X-ray scattering over normal tissues, distinguishing cells containing gold nanoparticles from cells without gold in X-ray scatter images. Our results suggest that this novel approach could enable the in vivo detection of tumors as small as a few millimeters in size.

Ethanol inhibits antigen presentation by dendritic cells.

Previous studies suggest that altered virus-specific T-cell responses observed during chronic ethanol exposure may be due to abnormal functioning of dendritic cells (DCs). Here we explored the effects of ethanol on exogenous antigen presentation by DCs. BALB/c, C57BL/6, and CBA/caj mice were fed ethanol or an isocaloric control diet for 8 weeks. The splenic DC population was expanded using an Flt3L expression plasmid via tail vein injection. DCs were purified and assessed for antigen presentation and processing and for peptide-major histocompatibility complex class I and II (MHCI and MHCII) formation on the cell surface. Interleukin-2 (IL-2) was measured as an indicator of antigen-specific T-cell activation by DCs in coculture. Antigen processing and peptide-MHCII complexes were evaluated by flow cytometry. We observed that ethanol not only suppressed allogeneic peptide presentation to T cells by DCs but also altered presentation of exogenous ovalbumin (OVA) peptide 323-339 to an OVA-specific DO11 T-cell line as well as to OVA-sensitized primary T cells. Smaller amounts of peptide-MHCII complexes were found on the DCs isolated from the spleens of ethanol-fed mice. In contrast to MHCII presentation, cross-presentation of exogenous OVA peptide via MHCI by DCs remained intact. More importantly, ethanol-exposed DCs had reduced B7-DC and enhanced ICOS-L (inhibitory) costimulatory molecule expression. Ethanol inhibits exogenous and allogeneic antigen presentation and affects the formation of peptide-MHCII complexes, as well as altering costimulatory molecule expression on the cell surface. Therefore, DC presentation of peptides in a favorable costimulatory protein environment is required to subsequently activate T cells and appears to be a critical target for the immunosuppressive effects of ethanol.

Chromosome numbers, nuclear DNA content, and polyploidy in Consolea (Cactaceae), an endemic cactus of the Caribbean Islands.

Polyploidy, an important mechanism of plant evolution, was investigated in Consolea, an endemic Caribbean opuntioid genus represented by nine subdioecious species with very narrow distributions, including species classified as rare or threatened. Standard chromosome counting and flow cytometric analyses were used to determine chromosome numbers and ploidy of each taxon. Compared to the base number (x = 11), the mitotic and meiotic counts indicated that there are seven hexaploid (2n = 66) and two octoploid species (2n = 88); no diploids were found. Histograms of intact nuclei confirmed that all species are polyploid, with C-DNA values ranging from 4.88-9.50 pg. The variation of DNA content was significantly higher for the octoploids than for the hexaploids. Male and female sexual morphs had similar DNA content, suggesting that there are no sex chromosomes. Cytomixis between cells and microsporocytes with no chromatin were observed. This provides a mechanism whereby gametes with variable chromosome numbers are produced, influencing reproduction and promoting speciation. In conclusion, C-DNA content and chromosome number separated Consolea species into two groups, which may correspond to two phylogenetic lineages or indicate that polyploidization occurred independently, with comparable effects on C-DNA content.

Subdioecy in Consolea spinosissima (Cactaceae): breeding system and embryological studies.

The breeding system and the embryology of Consolea spinosissima, a tree-like opuntioid endemic to Jamaica, were investigated. Morphological and embryological studies revealed that the species is subdioecious, with three sexual morphs present in the 150 × 120 m plot studied at Hellshire Hills, Jamaica. The female morph has pistillate flowers with open stigma lobes, no pollen grains, and sets fruit. The male morph has cryptic staminate flowers with closed stigma lobes, viable pollen grains, and a nonfunctional gynoecium that does not set seed. The weak hermaphrodite morph has low fruit set and "perfect" flowers that superficially resemble the functionally staminate flowers of the male morph. These perfect flowers reach anthesis with viable pollen grains, with no or only a few functional ovules, and with the style supporting pollen tube growth. Embryological studies showed that the critical stage for sex determination occurs earlier in pistillate than in staminate and perfect flowers. Anthers of pistillate flowers abort prior to microspore tetrad formation, whereas ovules of the staminate and perfect flowers degenerate after the complete maturation of the embryo sac. Based on flower structure and embryological data, we hypothesize that the ancestor of C. spinosissima is/was hermaphroditic.