Targeted detection of cancer cells during biopsy allows real-time diagnosis of pulmonary nodules.

BACKGROUND: The diagnostic yield of biopsies of solitary pulmonary nodules (SPNs) is low, particularly in sub-solid lesions. We developed a method (NIR-nCLE) to achieve cellular level cancer detection during biopsy by integrating (i) near-infrared (NIR) imaging using a cancer-targeted tracer (pafolacianine), and (ii) a flexible NIR confocal laser endomicroscopy (CLE) system that can fit within a biopsy needle. Our goal was to assess the diagnostic accuracy of NIR-nCLE ex vivo in SPNs. METHODS: Twenty patients with SPNs were preoperatively infused with pafolacianine. Following resection, specimens were inspected to identify the lesion of interest. NIR-nCLE imaging followed by tissue biopsy was performed within the lesion and in normal lung tissue. All imaging sequences (n = 115) were scored by 5 blinded raters on the presence of fluorescent cancer cells and compared to diagnoses by a thoracic pathologist. RESULTS: Most lesions (n = 15, 71%) were adenocarcinoma-spectrum malignancies, including 7 ground glass opacities (33%). Mean fluorescence intensity (MFI) by NIR-nCLE for tumor biopsy was 20.6 arbitrary units (A.U.) and mean MFI for normal lung was 6.4 A.U. (p < 0.001). Receiver operating characteristic analysis yielded a high area under the curve for MFI (AUC = 0.951). Blinded raters scored the NIR-nCLE sequences on the presence of fluorescent cancer cells with sensitivity and specificity of 98% and 97%, respectively. Overall diagnostic accuracy was 97%. The inter-observer agreement of the five raters was excellent (κ = 0.95). CONCLUSIONS: NIR-nCLE allows sensitive and specific detection of cancer cells in SPNs. This technology has far-reaching implications for diagnostic needle biopsies and intraprocedural decision-making.

Characterization of a Cryopreserved Split-Thickness Human Skin Allograft-TheraSkin.

OBJECTIVE: The purpose of this study was to examine the characteristics of a cryopreserved split-thickness skin allograft produced from donated human skin and compare it with fresh, unprocessed human split-thickness skin. BACKGROUND: Cutaneous wound healing is a complex and organized process, where the body re-establishes the integrity of the injured tissue. However, chronic wounds, such as diabetic or venous stasis ulcers, are difficult to manage and often require advanced biologics to facilitate healing. An ideal wound care product is able to directly influence wound healing by introducing biocompatible extracellular matrices, growth factors, and viable cells to the wound bed. MATERIALS AND METHODS: TheraSkin (processed by LifeNet Health, Virginia Beach, Virginia, and distributed by Soluble Systems, Newport News, Virginia) is a minimally manipulated, cryopreserved split-thickness human skin allograft, which contains natural extracellular matrices, native growth factors, and viable cells. The authors characterized TheraSkin in terms of the collagen and growth factor composition using ELISA, percentage of apoptotic cells using TUNEL analysis, and cellular viability using alamarBlue assay (Thermo Fisher Scientific, Waltham, Massachusetts), and compared these characteristics with fresh, unprocessed human split-thickness skin. RESULTS: It was found that the amount of the type I and type III collagen, as well as the ratio of type I to type III collagen in TheraSkin, is equivalent to fresh unprocessed human split-thickness skin. Similar quantities of vascular endothelial growth factor, insulinlike growth factor 1, fibroblast growth factor 2, and transforming growth factor ß1 were detected in TheraSkin and fresh human skin. The average percent of apoptotic cells was 34.3% and 3.1% for TheraSkin and fresh skin, respectively. CONCLUSIONS: Cellular viability was demonstrated in both TheraSkin and fresh skin.

Decellularization of human dermis using non-denaturing anionic detergent and endonuclease: a review.

Decellularized human dermis has been used for a number of clinical applications including wound healing, soft tissue reconstruction, and sports medicine procedures. A variety of methods exist to prepare this useful class of biomaterial. Here, we describe a decellularization technology (MatrACELL(®)) utilizing a non-denaturing anionic detergent, N-Lauroyl sarcosinate, and endonuclease, which was developed to remove potentially immunogenic material while retaining biomechanical properties. Effective decellularization was demonstrated by a residual DNA content of ≤4 ng/mg of wet weight which represented >97 % DNA removal compared to unprocessed dermis. Two millimeter thick MatrACELL processed human acellular dermal matrix (MH-ADM) exhibited average ultimate tensile load to failure of 635.4 ± 199.9 N and average suture retention strength of 134.9 ± 55.1 N. Using an in vivo mouse skin excisional model, MH-ADM was shown to be biocompatible and capable of supporting cellular and vascular in-growth. Finally, clinical studies of MH-ADM in variety of applications suggest it can be an appropriate scaffold for wound healing, soft tissue reconstruction, and soft tissue augmentation.

Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage.

The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (π(PG)), and the collagen network (CN) provides the restraining forces to counterbalance π(PG). Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-π(PG) relationship; 2), compute π(PG) and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on π(PG) in human aging. Extrafibrillar FCD (FCD(EF)) and π(PG) increased with bovine growth due to an increase in CN concentration, whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD(EF) and π(PG) decreased. The π(PG)-values were close to equilibrium stress (σ(EQ)) in all bovine and young human cartilage, but were only approximately half of σ(EQ) in old human cartilage. Depth-related variations in the strain, FCD(EF), π(PG), and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-π(PG) relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage.

Studying submicrosecond protein folding kinetics using a photolabile caging strategy and time-resolved photoacoustic calorimetry.

Kinetic measurement of protein folding is limited by the method used to trigger folding. Traditional methods, such as stopped flow, have a long mixing dead time and cannot be used to monitor fast folding processes. Here, we report a compound, 4-(bromomethyl)-6,7-dimethoxycoumarin, that can be used as a "photolabile cage" to study the early stages of protein folding. The folding process of a protein, RD1, including kinetics, enthalpy, and volume change, was studied by the combined use of a phototriggered caging strategy and time-resolved photoacoustic calorimetry. The cage caused unfolding of the photolabile protein, and then a pulse UV laser (∼10(-9) s) was used to break the cage, leaving the protein free to refold and allowing the resolving of two folding events on a nanosecond time scale. This strategy is especially good for monitoring fast folding proteins that cannot be studied by traditional methods.

A highly sensitive and dynamic immunofluorescent cytometric bead assay for the detection of HIV-1 p24.

Nucleic acid measurements are used to follow HIV-1 viral load in clinical applications while p24 ELISA is commonly used to monitor HIV-1 replication in research settings. Current ELISA assays are expensive and offer a narrow dynamic measurement range. This report describes a simple, sensitive and inexpensive bead-based assay offering a wide dynamic measurement range. This cytometric bead assay allows the detection of p24 concentrations over 4 orders of magnitude from less than 0.4pg to up to 20,000pgml(-1) in a volume of 50microl and can be combined with other measurements.

Comparison of Decellularized Cow and Human Bone for Engineering Bone Grafts with Human Induced Pluripotent Stem Cells.

IMPACT STATEMENT: Decellularized tissue matrices are popular as scaffolding materials for tissue engineering application. However, it is unclear whether interspecies differences in tissue parameters influence the quality of tissue grafts that are engineered using human stem cells. In this study, decellularized cow and human bone scaffolds were compared for engineering bone grafts using human induced pluripotent stem cell-derived mesodermal progenitor cells and despite minor differences in architecture and mass composition, both scaffolds equally support cell viability and tissue mineralization. Decellularized cow bone scaffolds therefore represent a suitable and more affordable alternative for engineering human bone grafts for basic and applied research.

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

INTRODUCTION: Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. METHODS AND RESULTS: Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. CONCLUSION: Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. STATEMENT OF SIGNIFICANCE: This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors.

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2-6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells.

Differentiation of rhesus monkey embryonic stem cells in three-dimensional collagen matrix.

During normal embryogenesis, embryonic stem cells (ESCs) reside in the context of complex three-dimensional tissue structures, in particular of extracellular matrices (ECMs), which determine cell migration, proliferation, and differentiation. Therefore, to study ESC differentiation in an in vivo-like microenvironment, three-dimensional culture systems are necessary. Here, we developed protocols for ESC cultures in three-dimensional systems consisting of collagen matrices (collagen gels and porous collagen sponges) to investigate the mechanisms of ESC differentiation as well as the formation of tissue-like structures. In collagen matrices, ESCs differentiate into neural, epithelial, and endothelial lineages. In this system, ESCs form various tissue-like structures. The abilities of ESCs to form such structures in two chemically similar but topologically different matrices are different. In particular, in collagen gels ESCs form gland-like circular structures, whereas in collagen sponges ESCs are scattered through the matrix and form aggregates. To mimic the in vivo situation further, we developed a protocol for co-cultures of ESCs with human dermal fibroblasts or keratinocytes in collagen matrixes. Co-culture with fibroblasts in collagen gel facilitates ESC differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule (NCAM), and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ESC differentiation into cells of an endothelial lineage expressing factor VIII. Thus, the developed protocols promote ESC differentiation into a particular lineage, accompanied by the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for directing ESC differentiation and the formation of organs and tissues.

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station.

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

Reconstruction of functional cortical-like tissues from neural stem and progenitor cells.

Neural stem and progenitor cells isolated from embryonic day 13 rat cerebral cortex were immobilized in three-dimensional type I collagen gels, and then the cell-collagen constructs were transferred to rotary wall vessel bioreactors and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor for up to 10 weeks. Remarkably, the collagen-entrapped cells formed a complex two-layered structure that emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The surface layer (layer I) composed primarily of proliferating neural progenitor cells (nestin(+), vimentin(+), and PCNA(+)) predominantly expressed functional neurotransmitter receptors for cholinergic and purinergic agonists while differentiating cells (TuJ1(+) and GFAP(+)) in the deeper layer (layer II) contained differentiated neurons and astrocytes and mainly responded to GABAergic and glutamatergic agonists and to veratridine, which activates voltage-dependent Na(+) channels. An active synaptic vesicle recycling was demonstrated by neuronal networks in the deeper layer using the endocytotic marker FM1-43. Cell polarization forming the characteristic two-layered structure was found to associate with the bFGF and FGF receptor signaling. These engineered functional tissue constructs have a potential use as tissue surrogates for drug screening and detection of environmental toxins, and in neural cell replacement therapy.

Cell-cell and cell-extracellular matrix interactions regulate embryonic stem cell differentiation.

Cell interactions with the extracellular matrix (ECM) play a critical role in their physiology. Here, we sought to determine the role of exogenous and endogenous ECM in the differentiation of nonhuman primate ESCs. We evaluated cell differentiation from expression of lineage gene mRNA and proteins using real-time polymerase chain reaction and immunohistochemistry. We found that ESCs that attached to and spread upon highly adhesive collagen do not differentiate efficiently, whereas on the less adhesive Matrigel, ESCs form aggregates and differentiate along mesoderm and especially endoderm lineages. To further decrease ESC attachment to the substrate, we cultured them either on nonadhesive agarose or in suspension. In both cases, ESCs formed aggregates and efficiently differentiated along endoderm and mesoderm lineages, most strikingly into cardiomyocytes. Aggregates formed by thus-differentiated ESCs started to beat with a frequency of 50-100 beats per minute and continued to beat for approximately a month. In spite of the presence of exogenous ECM, ESCs were dependent on endogenous ECM for their survival and differentiation, as the inhibition of endogenous collagen induced a gradual loss of ESCs and neither a simple matrix, such as type I collagen, nor the complex matrix Matrigel was able to rescue these cells. In conclusion, adhesiveness to various ECM and nonbiological substrates determines the differentiation of ESCs in such a way that efficient cell-cell aggregation, together with less efficient cell attachment and spreading, results in more efficient cell differentiation.

Complex extracellular matrices promote tissue-specific stem cell differentiation.

Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA-developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin-1 or collagen I, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular- and tubular-like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular- and glandular-like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue-stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.

Differential pathogenesis of primary CCR5-using human immunodeficiency virus type 1 isolates in ex vivo human lymphoid tissue.

In the course of human immunodeficiency virus (HIV) disease, CCR5-utilizing HIV type 1 (HIV-1) variants (R5), which typically transmit infection and dominate its early stages, persist in approximately half of the infected individuals (nonswitch virus patients), while in the other half (switch virus patients), viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. Here, we used a system of ex vivo tonsillar tissue to compare the pathogeneses of sequential primary R5 HIV-1 isolates from patients in these two categories. The absolute replicative capacities of HIV-1 isolates seemed to be controlled by tissue factors. In contrast, the replication level hierarchy among sequential isolates and the levels of CCR5(+) CD4(+) T-cell depletion caused by the R5 isolates seemed to be controlled by viral factors. R5 viruses isolated from nonswitch virus patients depleted more target cells than R5 viruses isolated from switch virus patients. The high depletion of CCR5(+) cells by HIV-1 isolates from nonswitch virus patients may explain the steady decline of CD4(+) T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value reflecting whether, in an infected individual, X4 HIV-1 will eventually dominate.

CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue by selective induction of CC-chemokines.

In infected individuals, human immunodeficiency virus type 1 (HIV-1) exist as a "swarm" of quasi species compartmentalized in tissues where individual viral variants may interact locally. We have used human lymphoid tissue, where the critical events of HIV disease occur, to study local interactions in model HIV-1 binary swarms ex vivo. We infected tissue blocks with binary mixtures consisting either of CCR5-dependent and CXCR4-dependent variants or of 2 dual-tropic HIV-1 variants, of which one is skewed to utilization of CXCR4 and the other of CCR5. HIV-1 variants that use CXCR4 suppress replication of CCR5-dependent HIV-1 variants, whereas CCR5-dependent HIV-1 variants do not affect replication of CXCR4-dependent HIV-1. CC-chemokines that inhibit replication of CCR5-dependent HIV-1 variants were up-regulated by CXCR4-dependent HIV-1, thus possibly contributing to this suppression. Tissue-specific chemokine/cytokine network modulations triggered by individual HIV-1 variants may be an important mechanism of local interactions among HIV-1 quasi species in infected tissue.

A growth mixture theory for cartilage with application to growth-related experiments on cartilage explants.

In this paper, we present a growth mixture model for cartilage. The main features of this model are illustrated in a simple equilibrium boundary-value problem that is chosen to illustrate how a mechanical theory of cartilage growth may be applied to growth-related experiments on cartilage explants. The cartilage growth mixture model describes the independent growth of the proteoglycan and collagen constituents due to volumetric mass deposition, which leads to the remodeling of the composition and the mechanical properties of the solid matrix. The model developed here also describes how the material constants of the collagen constituent depend on a scalar parameter that may change over time (e.g., crosslink density); this leads to a remodeling of the structural and mechanical properties of the collagen constituent. The equilibrium boundary-value problem that describes the changes observed in cartilage explants harvested at different stages of a growth or a degenerative process is formulated. This boundary-value problem is solved using existing experimental data for developing bovine cartilage explants harvested at three developmental stages. The solution of the boundary-value problem in conjunction with existing experimental data suggest the types of experimental studies that need to be conducted in the future to determine model parameters and to further refine the model.

Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo.

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to productively infect human tonsil tissue fragments in the absence of exogenous stimulation. The majority of viral antigen-expressing cells were CD4(+) T lymphocytes expressing a nonnaive phenotype, while CD8(+) T cells were efficiently infected only with HHV-6A. Accordingly, HHV-6A infection resulted in the depletion of both CD4(+) and CD8(+) T cells, whereas in HHV-6B-infected tissue CD4(+) T cells were predominantly depleted. The expression of different cellular antigens was dramatically altered in HHV-6-infected tissues: whereas CD4 was upregulated, both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. However, CD3 downmodulation was restricted to infected cells, while the loss of CD46 expression was generalized. Moreover, HHV-6 infection markedly enhanced the production of the CC chemokine RANTES, whereas other cytokines and chemokines were only marginally affected. These results provide the first evidence, in a physiologically relevant study model, that HHV-6 can severely affect the physiology of secondary lymphoid organs through direct infection of T lymphocytes and modulation of key membrane receptors and chemokines.

Isolation and clonal analysis of human epidermal keratinocyte stem cells in long-term culture.

We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.