RESUMO
Breastfeeding has been regarded first and foremost as a means of nutrition for infants, providing essential components for their unique growth and developmental requirements. However, breast milk is also rich in immunologic factors, highlighting its importance as a mediator of protection. In accordance with its evolutionary origin, the mammary gland offers via the breastfeeding route continuation of the maternal to infant immunologic support established in utero. At birth, the infant's immune system is immature, and although it was exposed to the maternal microbial flora during pregnancy, it experiences an abrupt change in its microbial environment during and after birth, which is challenging and renders the infant highly susceptible to infection. Active and passive immunity protects the infant via breast milk, which is rich in immunoglobulins, lactoferrin, lysozyme, cytokines, and numerous other immunologic factors, including maternal leukocytes. Breast milk leukocytes provide active immunity and promote development of immunocompetence in the infant. Additionally, it has been speculated that they play a role in the protection of the mammary gland from infection. Leukocytes are thought to exert these functions via phagocytosis, secretion of antimicrobial factors and/or antigen presentation in both the mammary gland and the gastrointestinal tract of the infant, and also in other infant tissues, where they are transported via the systemic circulation. Recently, it has been demonstrated that breast milk leukocytes respond dynamically to maternal as well as infant infections, and are fewer in nonexclusively compared with exclusively breastfeeding dyads, further emphasizing their importance for both the mother and infant. This review summarizes the current knowledge of human milk leukocytes and factors influencing them, and presents recent novel findings supporting their potential as a diagnostic marker for infections of the lactating breast and of the breastfed infant.
Assuntos
Doenças Mamárias/prevenção & controle , Aleitamento Materno , Infecções , Lactação/imunologia , Leucócitos/metabolismo , Glândulas Mamárias Humanas/imunologia , Leite Humano/metabolismo , Animais , Doenças Mamárias/imunologia , Humanos , Sistema Imunitário/crescimento & desenvolvimento , Lactente , Infecções/imunologia , MãesRESUMO
MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column-based phenol-free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area.
Assuntos
Lactação/metabolismo , MicroRNAs/isolamento & purificação , RNA/isolamento & purificação , Mama/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Lactente , Metabolismo dos Lipídeos/genética , MicroRNAs/biossíntese , MicroRNAs/química , Leite Humano/química , RNA/químicaAssuntos
Aleitamento Materno , Leite Humano/citologia , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Contagem de Células , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Citometria de Fluxo , Humanos , Lactente , Recém-Nascido , Lactação/fisiologia , Camundongos , Modelos BiológicosRESUMO
This work studies a model for milk transport through lactating human breast ducts and describes mathematically the mass transfer from alveolar sacs through the mammary ducts to the nipple. In this model, both the phenomena of diffusion in the sacs and conventional flow in ducts have been considered. The ensuing analysis reveals that there is an optimal range of bifurcation numbers leading to the easiest milk flow based on the minimum flow resistance. This model formulates certain difficult-to-measure values like diameter of the alveolar sacs and the total length of the milk path as a function of easy-to-measure properties such as milk fluid properties and macroscopic measurements of the breast. Alveolar dimensions from breast tissues of six lactating women are measured and reported in this paper. The theoretically calculated alveoli diameters for optimum milk flow (as a function of bifurcation numbers) show excellent match with our biological data on alveolar dimensions. Also, the mathematical model indicates that for minimum milk flow resistance the glandular tissue must be within a short distance from the base of the nipple, an observation that matches well with the latest anatomical and physiological research.
Assuntos
Lactação , Glândulas Mamárias Humanas/fisiologia , Modelos Biológicos , Difusão , Feminino , Humanos , Hidrodinâmica , Glândulas Mamárias Humanas/citologia , Leite Humano/metabolismoRESUMO
Glioblastoma is the most common and most aggressive primary brain malignancy. The current initial standard of care consists of maximal safe surgical resection followed by radical radiotherapy and adjuvant temozolomide. Despite optimal therapy, median survival is ~15 months from diagnosis in molecularly unselected patients, and <6 months for patients with recurrent disease. Therefore, clinical treatments are currently palliative, not curative. Collectively, current knowledge suggests that the continued tumor growth and recurrence is in part due to the presence of glioma stem-like cells, which display self-renewal and tumorigenic potential. They differ from their more differentiated progeny, as they are more resistant to current treatments. Recurrent disease may be a consequence of the enhancement and/or gain of stem cell-like characteristics during disease progression, together with preferential death of more differentiated tumor cells during treatment, signifying that the cancer stem cell phenotype is a crucial therapeutic target. The limited knowledge of the characteristics of these cells and their response to current clinical treatments warrants intensive investigation with the aim to improve patient survival and/or develop a cure for this disease.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Antígenos CD/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Peptídeos/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidoresRESUMO
Human milk is the optimal source of nutrition for the nursing infant. Classically, the nutrients (water, protein, lipid, carbohydrate, vitamins, and minerals) were studied as the critical components of milk serving the growth needs of the infant for optimum growth. However, human milk contains factors other than the classically defined nutrients for which researchers are investigating potential roles in infant and maternal health, development, and well-being. The symposium addressed some of the exciting factors being studied, including microbes and maternal cells found within milk. Drs. Michelle McGuire and Juan M. Rodríguez addressed the presence of a bacterial community in human milk produced by healthy and mastitic mothers, potential sources of those bacteria, and the impact of milk-derived bacteria on the nursing infant. Drs. Donna Geddes, Peter Hartmann, and Foteini Hassiotou discussed the potential importance of maternal cells. For years, immune cells were known to be present in human milk, but recent evidence suggests that their impact is as much on the infant as on the health of the lactating mammary gland. Finally, the existence of highly plastic stem cells in human milk opens doors for previously unforeseen developmental "training" of the nursing infant.
Assuntos
Aleitamento Materno , Leite Humano/química , Leite Humano/microbiologia , Congressos como Assunto , Feminino , Humanos , Lactente , Microbiota , Leite Humano/imunologia , Estado Nutricional , ProbióticosRESUMO
Breast milk contains bioactive molecules that provide a multitude of immunologic, developmental and nutritional benefits to the infant. Less attention has been placed on the cellular nature of breast milk, which contains thousands to millions of maternal cells in every milliliter that the infant ingests. What are the properties and roles of these cells? Most studies have examined breast milk cells from an immunologic perspective, focusing specifically on the leukocytes, mainly in the early postpartum period. In the past decade, research has taken a multidimensional approach to investigating the cells of human milk. Technologic advances in single cell analysis and imaging have aided this work, which has resulted in the breakthrough discovery of stem cells in breast milk with multilineage potential that are transferred to the offspring during breastfeeding. This has generated numerous implications for both infant and maternal health and regenerative medicine. This review summarizes the latest knowledge on breast milk stem cells, and discusses their known in vitro and in vivo attributes as well as potential functions and applications.
Assuntos
Aleitamento Materno , Leite Humano/citologia , Células-Tronco/citologia , Feminino , Humanos , Lactação , Período Pós-PartoRESUMO
This work studies a simple model for milk transport through lactating human breast ducts, and describes mathematically the mass transfer from alveolar sacs through the mammary ducts to the nipple. In this model both the phenomena of diffusion in the sacs and conventional flow in ducts have been considered. The ensuing analysis reveals that there is an optimal range of bifurcation numbers leading to the easiest milk flow based on the minimum flow resistance. This model formulates certain difficult-to-measure values like diameter of the alveolar sacs, and the total length of the milk path as a function of easy-to-measure properties such as milk fluid properties and macroscopic measurements of the breast. Alveolar dimensions from breast tissues of six lactating women are measured and reported in this paper. The theoretically calculated alveoli diameters for optimum milk flow (as a function of bifurcation numbers) show excellent match with our biological data on alveolar dimensions. Also, the mathematical model indicates that for minimum milk flow resistance the glandular tissue must be within a short distance from the base of the nipple, an observation that matches well with the latest anatomical and physiological research.
Assuntos
Glândulas Mamárias Humanas/fisiologia , Modelos Biológicos , Algoritmos , Animais , Feminino , Humanos , Hidrodinâmica , Lactação , Glândulas Mamárias Humanas/anatomia & histologia , Leite Humano/metabolismo , Mamilos/anatomia & histologia , Mamilos/diagnóstico por imagem , Mamilos/fisiologia , UltrassonografiaRESUMO
Large inter- and intra-individual variations exist in breastmilk composition, yet factors associated with these variations in the short-term are not well understood. In this study, the effects of breastfeeding on breastmilk cellular and biochemical content were examined. Serial breastmilk samples (â¼5 mL) were collected from both breasts of breastfeeding women before and immediately after the first morning breastfeed, and then at 30-minute intervals for up to 3 hours post-feed on 2-4 mornings per participant. The infant fed from one breast only at each feed. Effects of pump versus hand expression for samples were evaluated. A consistent response pattern of breastmilk cell and fat contents to breastmilk removal was observed. Maximum fat and cell levels were obtained 30 minutes post-feed (P<0.01), with up to 8-fold increase in fat and 12-fold increase in cell content compared to the pre-feed values, and then they gradually decreased. Breastmilk cell viability and protein concentration did not change with feeding (P>0.05), although large intra-individual variability was noted for protein. Expression mode for samples did not influence breastmilk composition (P>0.05). It is concluded that breastmilk fat content, and thus breast fullness, is closely associated with breastmilk cell content. This will now form the basis for standardization of sampling protocols in lactation studies and investigation of the mechanisms of milk synthesis and cell movement into breastmilk. Moreover, these findings generate new avenues for clinical interventions exploring growth and survival benefits conferred to preterm infants by providing the highest in fat and cells milk obtained at 30 min post-expression.
Assuntos
Aleitamento Materno , Lactação/fisiologia , Lipídeos/análise , Proteínas do Leite/análise , Leite Humano , Adulto , Extração de Leite/métodos , Sobrevivência Celular , Feminino , Humanos , Lactente , Recém-Nascido , Leite Humano/química , Leite Humano/citologia , Fatores de TempoRESUMO
Breast tumors are heterogeneous including cells with stem cell properties and more differentiated cells. This heterogeneity is reflected into the molecular breast cancer subtypes. Breast cancer stem cells are resistant to chemotherapy, thus recent efforts are focusing on identifying treatments that shift them toward a more differentiated phenotype, making them more susceptible to chemotherapy. We examined whether the drug cisplatin induces differentiation in breast cancer cell lines that represent different breast cancer subtypes. We used three cell lines representing triple-negative breast cancers, BT-549 and MDA-MB-231 (claudin-low), and MDA-MB-468 (basal-like), along with estrogen and progesterone receptor positive MCF-7 cells (luminal). Cisplatin was applied at 2.5, 5, 10, and 20 µM, and cell viability and proliferation were measured using MTS and BrdU assays, respectively. The effect of cisplatin on the cellular hierarchy was examined by flow cytometry, immunofluorescence and qRT-PCR. Cisplatin treatment of 10 and 20 µM reduced cell viability by 36-51% and proliferation capacity by 36-67%. Treatment with cisplatin resulted in 12-67% down-regulation of stem cell markers (CD49f, SSEA4) and 10-130% up-regulation of differentiation markers (CK18, SMA, ß-tubulin). At the mRNA level, CD49f was down-regulated whilst ß-tubulin was up-regulated in the claudin-low cell lines. SSEA4 protein expression decreased upon cisplatin treatment, but SSEA4 mRNA expression increased indicating a differential regulation of cisplatin at the post-transcriptional level. It is concluded that cisplatin reduces breast cancer cell survival and induces differentiation of stem/progenitor cell subpopulations within breast cancer cell lines. These effects indicate the potential of this drug to target specific chemotherapy-resistant cells within a tumor.
RESUMO
Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor, and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast (from non-pregnant, non-lactating women) showed minimal OCT4 nuclear expression (0.9%). However, this increased in the normal lactating breast (11.4%), with further increase in lactating adenomas, lactating carcinomas, and pregnancy-associated breast cancer (30.7-48.3%). OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs) demonstrated upregulation of OCT4, SOX2, and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both in vitro and in vivo. Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the normal remodeling of the breast toward a milk-secretory organ during pregnancy and lactation. Deregulation of this gene network either within or outside pregnancy and lactation may lead to aberrant breast cell proliferation and malignant transformation, suggesting a role of these genes in both normal lactation and breast oncogenesis.
RESUMO
Reflecting millions of years of adaptation and optimization, milk is unique to the species that produces it and for the young of which it is intended, with large variations in both lactation strategies and milk composition existing among different mammalian species. Despite this, milk has the consistent function of providing nourishment, protection, and developmental programming to the young, with short- and long-term effects. Among its components that confer these functions, breast milk contains maternal cells, from leukocytes to epithelial cells of various developmental stages that include stem cells, progenitor cells, lactocytes, and myoepithelial cells. Although in the first 150 years since their discovery, breast milk cells were mostly studied for their morphological traits, technological advances in the last decade have allowed characterization of breast milk cell types at the protein and messenger RNA levels. This is now paving the way for investigation of the functions of these cells in the breastfed infant and the use of breast milk as a tool to understand the normal biology of the breast and its pathologies. This review summarizes the current knowledge of breast milk cellular heterogeneity and discusses future prospects and potential applications.
Assuntos
Células Epiteliais/citologia , Leite Humano/citologia , Células Espumosas/citologia , Humanos , Leucócitos/citologia , Proteínas do Leite/biossíntese , Células-Tronco/citologia , Fatores de TempoRESUMO
Breastmilk protects infants against infections; however, specific responses of breastmilk immune factors to different infections of either the mother or the infant are not well understood. Here, we examined the baseline range of breastmilk leukocytes and immunomodulatory biomolecules in healthy mother/infant dyads and how they are influenced by infections of the dyad. Consistent with a greater immunological need in the early postpartum period, colostrum contained considerable numbers of leukocytes (13-70% out of total cells) and high levels of immunoglobulins and lactoferrin. Within the first 1-2 weeks postpartum, leukocyte numbers decreased significantly to a low baseline level in mature breastmilk (0-2%) (P<0.001). This baseline level was maintained throughout lactation unless the mother and/or her infant became infected, when leukocyte numbers significantly increased up to 94% leukocytes out of total cells (P<0.001). Upon recovery from the infection, baseline values were restored. The strong leukocyte response to infection was accompanied by a more variable humoral immune response. Exclusive breastfeeding was associated with a greater baseline level of leukocytes in mature breastmilk. Collectively, our results suggest a strong association between the health status of the mother/infant dyad and breastmilk leukocyte levels. This could be used as a diagnostic tool for assessment of the health status of the lactating breast as well as the breastfeeding mother and infant.
RESUMO
Mammary glands are unique to mammals, with the specific function of synthesizing, secreting, and delivering milk to the newborn. Given this function, it is only during a pregnancy/lactation cycle that the gland reaches a mature developmental state via hormonal influences at the cellular level that effect drastic modifications in the micro- and macro-anatomy of the gland, resulting in remodeling of the gland into a milk-secretory organ. Pubertal and post-pubertal development of the breast in females aids in preparing it to assume a functional state during pregnancy and lactation. Remarkably, this organ has the capacity to regress to a resting state upon cessation of lactation, and then undergo the same cycle of expansion and regression again in subsequent pregnancies during reproductive life. This plasticity suggests tight hormonal regulation, which is paramount for the normal function of the gland. This review presents the current status of knowledge of the normal macro- and micro-anatomy of the human mammary gland and the distinct changes it undergoes during the key developmental stages that characterize it, from embryonic life through to post-menopausal age. In addition, it discusses recent advances in our understanding of the normal function of the breast during lactation, with special reference to breastmilk, its composition, and how it can be utilized as a tool to advance knowledge on normal and aberrant breast development and function. Finally, anatomical and molecular traits associated with aberrant expansion of the breast are discussed to set the basis for future comparisons that may illuminate the origin of breast cancer.
Assuntos
Lactação/fisiologia , Glândulas Mamárias Humanas/anatomia & histologia , Glândulas Mamárias Humanas/fisiologia , Gravidez/fisiologia , Feminino , Humanos , Glândulas Mamárias Humanas/embriologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Pós-Menopausa , PuberdadeRESUMO
The pregnancy-lactation cycle (PLC) is a period in which the breast is transformed from a less-developed, nonfunctional organ into a mature, milk-producing gland that has evolved to meet the nutritional, developmental, and immune protection needs of the newborn. Cessation of lactation initiates a process whereby the breast reverts to a resting state until the next pregnancy. Changes during this period permanently alter the morphology and molecular characteristics of the breast (molecular histology) and produce important, yet poorly understood, effects on breast cancer risk. To provide a state-of-the-science summary of this topic, the National Cancer Institute invited a multidisciplinary group of experts to participate in a workshop in Rockville, Maryland, on March 2, 2012. Topics discussed included: 1) the epidemiology of the PLC in relation to breast cancer risk, 2) breast milk as a biospecimen for molecular epidemiological and translational research, and 3) use of animal models to gain mechanistic insights into the effects of the PLC on breast carcinogenesis. This report summarizes conclusions of the workshop, proposes avenues for future research on the PLC and its relationship with breast cancer risk, and identifies opportunities to translate this knowledge to improve breast cancer outcomes.
Assuntos
Aleitamento Materno , Neoplasias da Mama/prevenção & controle , Mama/anatomia & histologia , Lactação , Leite Humano , Gravidez , Prevenção Primária , Adulto , Animais , Mama/fisiologia , Congressos como Assunto , Modelos Animais de Doenças , Medicina Baseada em Evidências , Feminino , Humanos , Comunicação Interdisciplinar , Leite Humano/química , Leite Humano/citologia , Paridade , Período Pós-Parto , Fatores de RiscoRESUMO
The mammary gland undergoes significant remodeling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here, we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4, SOX2, NANOG, known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts, a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium, they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore, hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer, with potential use in bioengineering and tissue regeneration.
Assuntos
Linhagem da Célula/fisiologia , Leite Humano/citologia , Células-Tronco Pluripotentes/citologia , Adulto , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Alimentadoras , Feminino , Fibroblastos , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Lactação , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Leaf dry mass per area (LMA) is a composite parameter relating to a suite of structural traits that have the potential to influence photosynthesis. However, the extent to which each of these traits contributes to variation in LMA and photosynthetic rates is not well understood, especially at the high end of the LMA spectrum. In this study, the genus Banksia (Proteaceae) was chosen as a model group, and key structural traits such as LMA, leaf thickness, and density were measured in 49 species. Based on the leaf trait variation obtained, a subset of 18 species displaying a wide range in LMA of 134-507 g m(-2) was selected for analyses of relationships between leaf structural and photosynthetic characteristics. High LMA was associated with more structural tissue, lower mass-based chlorophyll and nitrogen concentrations, and therefore lower mass-based photosynthesis. In contrast, area-based photosynthesis did not correlate with LMA, despite mesophyll volume per area increasing with increases in LMA. Photosynthetic rate per unit mesophyll volume declined with increasing LMA, which is possibly associated with structural limitations and, to a lesser extent, with lower nitrogen allocation. Mesophyll cell wall thickness significantly increased with LMA, which would contribute to lower mesophyll conductance at high LMA. Photosynthetic nitrogen use efficiency and the nitrogen allocation to Rubisco and thylakoids tended to decrease at high LMA. The interplay between anatomy and physiology renders area-based photosynthesis independent of LMA in Banksia species.
Assuntos
Fotossíntese , Proteaceae/química , Proteaceae/metabolismo , Locos de Características Quantitativas , Clorofila/metabolismo , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteaceae/genéticaRESUMO
Stomata arranged in crypts with trichomes are commonly considered to be adaptations to aridity due to the additional diffusion resistance associated with this arrangement; however, information on the effect of crypts on gas exchange, relative to stomata, is sparse. In this study, three-dimensional Finite Element models of encrypted stomata were generated using commercial Computational Fluid Dynamics software. The models were based on crypt and stomatal architectural characteristics of the species Banksia ilicifolia, examined microscopically, and variations thereof. In leaves with open or partially closed stomata, crypts reduced transpiration by less than 15% compared with nonencrypted, superficially positioned stomata. A larger effect of crypts was found only in models with unrealistically high stomatal conductances. Trichomes inside the crypt had virtually no influence on transpiration. Crypt conductance varied with stomatal conductance, boundary layer conductance, and ambient relative humidity, as these factors modified the three-dimensional diffusion patterns inside crypts. It was concluded that it is unlikely that the primary function of crypts and crypt trichomes is to reduce transpiration.