Both Manuka and Non-Manuka Honey Types Inhibit Antibiotic Resistant Wound-Infecting Bacteria.

Postoperative infections are a major concern in United States hospitals, accounting for roughly 20% of all hospital-acquired infections yearly. Wound-infecting bacteria, in particular, have a high rate of drug resistance (up to 65%), creating life-threatening complications. Manuka honey, native to New Zealand, has been FDA-approved for wound treatment in the United States after studies demonstrated its ability to inhibit a variety of bacterial species and facilitate wound healing. The aim of this study was to identify alternative (non-manuka) honey types that can be specifically used against antibiotic resistance bacteria in wound infections. We utilized a honey-plate method to measure the minimum inhibitory concentration (MIC) of honey to avoid the limitations of agar diffusion, where large, nonpolar polyphenols (which will not diffuse efficiently) play an important role in bioactivity. This study demonstrated that there are several alternative (non-manuka) honey types, particularly fresh raw Arkansas wildflower honeys, that comparably inhibit the growth of the antibiotic-resistant bacterial species specifically implicated in wound infections. Concentrations of 10-30% honey inhibited the growth of the highly antibiotic-resistant organisms colloquially referred to as "superbugs", which the WHO declared in 2017 to be in critical need of new antibiotics. There was no statistical difference between manuka honey and fresh summer Arkansas wildflower honey in overall bacterial inhibition. These results could transform wound care in the United States, where manuka honey can be expensive and difficult to obtain and where antibiotic resistance remains a troubling concern for wound treatment.

Contribution of Deep-Learning Techniques Toward Fighting COVID-19: A Bibliometric Analysis of Scholarly Production During 2020.

COVID-19 has dramatically affected various aspects of human society with worldwide repercussions. Firstly, a serious public health issue has been generated, resulting in millions of deaths. Also, the global economy, social coexistence, psychological status, mental health, and the human-environment relationship/dynamics have been seriously affected. Indeed, abrupt changes in our daily lives have been enforced, starting with a mandatory quarantine and the application of biosafety measures. Due to the magnitude of these effects, research efforts from different fields were rapidly concentrated around the current pandemic to mitigate its impact. Among these fields, Artificial Intelligence (AI) and Deep Learning (DL) have supported many research papers to help combat COVID-19. The present work addresses a bibliometric analysis of this scholarly production during 2020. Specifically, we analyse quantitative and qualitative indicators that give us insights into the factors that have allowed papers to reach a significant impact on traditional metrics and alternative ones registered in social networks, digital mainstream media, and public policy documents. In this regard, we study the correlations between these different metrics and attributes. Finally, we analyze how the last DL advances have been exploited in the context of the COVID-19 situation.

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application.

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human ß-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

Establishment, Genetic Diversity, and Habitat Suitability of Aedes albopictus Populations from Ecuador.

Aedes albopictus, also known as the tiger mosquito, is widespread worldwide across tropical, subtropical, and temperate regions. This insect is associated with the transmission of several vector-borne diseases, and, as such, monitoring its distribution is highly important for public health. In Ecuador, Ae. albopictus was first reported in 2017 in Guayaquil. Since then, the vector has been identified in the Northeastern lowlands and the Amazon basin. This study aims to determine the genetic diversity of Ecuadorian populations of Ae. albopictus through the analysis of the mitochondrial gene COI and to describe the potential distribution areas of this species within the country. The genetic diversity was determined by combining phylogenetic and population genetics analyses of five localities in Ecuador. Results showed two haplotypes in the Ecuadorian populations of Ae. albopictus. Haplotype 1 (H1) was found in the coastal and Amazon individuals, while haplotype 2 (H2) was only found in the three northeastern lowlands sites. In a worldwide context, H1 is the most widespread in 21 countries with temperate and tropical habitats. In contrast, H2 distribution is limited to five countries in tropical regions, suggesting fewer adaptation traits. Our prediction model showed a suitable habitat for Ae. albopictus in all regions (coastal, Amazon basin, and Andean lowland regions and the Galápagos Islands) of Ecuador. Hence, understanding different aspects of the vector can help us implement better control strategies for surveillance and vectorial control in Ecuador.

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the COVID-19 pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human {beta}-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values [≤] 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct [≤] 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

Determinants of Aedes mosquito density as an indicator of arbovirus transmission risk in three sites affected by co-circulation of globally spreading arboviruses in Colombia, Ecuador and Argentina.

BACKGROUND: The global impact of Zika virus in Latin America has drawn renewed attention to circulating mosquito-borne viruses in this region, such as dengue and chikungunya. Our objective was to assess socio-ecological factors associated with Aedes mosquito vector density as a measure of arbovirus transmission risk in three cities of potentially recent Zika virus introduction: Ibagué, Colombia; Manta, Ecuador; and Posadas, Argentina, in order to inform disease mitigation strategies. METHODS: We sampled Aedes mosquito populations in a total of 1086 households, using indoor and peridomestic mosquito collection methods, including light traps, resting traps, traps equipped with chemical attractant and aspirators. For each sampled household, we collected socio-economic data using structured questionnaires and data on microenvironmental conditions using iButton data loggers. RESULTS: A total of 3230 female Aedes mosquitoes were collected, of which 99.8% were Aedes aegypti and 0.2% were Aedes albopictus. Mean female Aedes mosquito density per household was 1.71 (standard deviation: 2.84). We used mixed-effects generalized linear Poisson regression analyses to identify predictors of Aedes density, using month, neighborhood and country as random-effects variables. Across study sites, the number of household occupants [incidence rate ratio (IRR): 1.08, 95% confidence interval (CI): 1.01-1.14], presence of entry points for mosquitoes into the household (IRR: 1.51, 95% CI: 1.30-1.76) and presence of decorative vegetation (IRR: 1.52, 95% CI: 1.22-1.88) were associated with higher Aedes density; while being in the highest wealth tertile of household wealth (IRR: 0.78, 95% CI: 0.66-0.92), knowledge of how arboviruses are transmitted (IRR: 0.94, 95% CI: 0.89-1.00) and regular emptying of water containers by occupants (IRR: 0.79, 95% CI: 0.67-0.92) were associated with lower Aedes density. CONCLUSIONS: Our study addresses the complexities of arbovirus vectors of global significance at the interface between human and mosquito populations. Our results point to several predictors of Aedes mosquito vector density in countries with co-circulation of multiple Aedes-borne viruses, and point to modifiable risk factors that may be useful for disease prevention and control.

Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the direct use of samples with an RNase inhibitor. Direct, extraction-free use of primary samples plus the RNase inhibitor produced diagnostic values of 100 % sensitivity and specificity compared to standard protocols, and these findings were validated in a second, independent laboratory.

Two Haplotypes of Aedes aegypti Detected by ND4 Mitochondrial Marker in Three Regions of Ecuador.

Aedes aegypti, also known as the yellow fever mosquito, is the main vector of several arboviruses. In Ecuador, dengue and chikungunya are the most prevalent mosquito-borne diseases. Hence, there is a need to understand the population dynamics and genetic structure of the vector in tropical areas for a better approach towards effective vector control programs. This study aimed to assess the genetic diversity of Ae. aegypti, through the analyses of the mitochondrial gene ND4, using a combination of phylogenetic and population genetic structure from 17 sites in Ecuador. Results showed two haplotypes in the Ecuadorian populations of Ae. aegypti. Haplotype 1 was closely related to Ae. aegypti reported from America, Asia, and West Africa. Haplotype 2 was only related to samples from America. The sampled vectors from the diverse localities showed low nucleotide diversity (π = 0-0.01685) and genetic differentiation (FST = 0.152). AMOVA analyses indicated that most of the variation (85-91%) occurred within populations, suggesting that geographical barriers have little effect on the genetic structure of Ecuadorian populations of Ae. aegypti. These results agree with the one main population (K = 1) detected by Structure. Vector genetic identity may be a key factor in the planning of vector control strategies.

Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling.

To address the issues of redundancy and specificity of chemokines and their receptors in lymphocyte biology, we investigated the expression of CC chemokine receptors CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4 and responses to their ligands on memory and naive, CD4 and CD8 human T cells, both freshly isolated and after short term activation in vitro. Activation through CD3 for 3 days had the most dramatic effects on the expression of CXCR3, which was up-regulated and functional on all T cell populations including naive CD4 cells. In contrast, the effects of short term activation on expression of other chemokine receptors was modest, and expression of CCR2, CCR3, and CCR5 on CD4 cells was restricted to memory subsets. In general, patterns of chemotaxis in the resting cells and calcium responses in the activated cells corresponded to the patterns of receptor expression among T cell subsets. In contrast, the pattern of calcium signaling among subsets of freshly isolated cells did not show a simple correlation with receptor expression, so the propensity to produce a global rise in the intracellular calcium concentration differed among the various receptors within a given T cell subset and for an individual receptor depending on the cell where it was expressed. Our data suggest that individual chemokine receptors and their ligands function on T cells at different stages of T cell activation/differentiation, with CXCR3 of particular importance on newly activated cells, and demonstrate T cell subset-specific and activation state-specific responses to chemokines that are achieved by regulating receptor signaling as well as receptor expression.

Identification of surface molecules associated with physiologic activation of eosinophils. Application of whole-blood flow cytometry to eosinophils.

Activation is central to the eosinophil's functional role as an immune responder cell. To evaluate such activation in cells freshly isolated from peripheral blood, a method for whole-blood immunostaining and flow cytometry-based eosinophil selection was developed. Simultaneous comparison of purified eosinophils and whole-blood cells revealed significant differences in the levels of expression of various surface molecules, which suggested that the purification process activated the eosinophils. Subsequent analyses were conducted with the whole-blood assay. When eosinophils from helminth-infected persons (n = 18) were compared with those from normal individuals (n = 10), the early activation marker CD69 was found to be significantly increased (geometric mean (GM) = 4.3 vs. 1.0%, p = 0.04). The granulocyte activation marker CD66 was also up-regulated on eosinphils from helminth patients (GM = 53.3 vs. 31.0%, p = 0.044), as was the tetraspan family molecule CD81 (TAPA-1; GM = 79.4 vs. 48.2%, p = 0.02). Conversely, in vivo CD23 (FcepsilonRII) expression on eosinophils was decreased in the presence of parasitic infection (GM = 0.9 vs. 5.7%, p = 0.02). Expression of the eosinophil surface molecules CD69, CD81, and CD23 was significantly enhanced after cytokine stimulation in vitro with IL-3 or GM-CSF. In vivo, specific anthelmintic therapy resulted in decreased CD66 and CD25 expression (p < 0.05 compared with pretreatment) to levels approaching those seen in uninfected normal individuals. These findings indicate the dynamic nature of eosinophil surface molecules and demonstrate an important role for whole-blood staining in developing an understanding of the nature of eosinophil activation and of their role in inflammatory reactions to helminth parasites.

Functional studies examining the subpopulation of human B lymphocytes responding to high molecular weight B cell growth factor.

Mature human B lymphocytes perform many functions including antibody secretion, Ag presentation, preservation of memory for Ag, and lymphokine secretion. Individual resting B cells receive multiple sequential signals that determine the function(s) that will be performed by those cells. Activation signals such as Ag or Staphylococcus aureus Cowan I (Sac) stimulate overlapping but different subpopulations of B cells. After activation, B cells may be induced to proliferate by a variety of B cell growth factors (BCGF) including IL-2, IL-4, TNF-alpha, low molecular weight BCGF (LMW-BCGF), and high molecular weight BCGF (HMW-BCGF). Little information exists to explain why so many different BCGFs are involved with human B cell proliferation. The current studies were designed to examine the role HMW-BCGF plays in selecting B cells for particular functions. HMW-BCGF but not LMW-BCGF was found to inhibit Ig secretion when it was included in culture with Sac-activated B cells and B cell differentiation factors (BCDFs) including IL-6. Sorting resting B lymphocytes into surface IgD+ and IgD- populations and then stimulating each population with anti-mu revealed that the cells most responsive to HMW-BCGF resided in the surface IgD- sorted population. Sorting activated B lymphocytes into BA5 (HMW-BCGFR)+ and BA5- populations revealed that BA5+ B cells stimulated with BCDF (in the absence of HMW-BCGF) produced predominantly IgG, whereas the BA5- population produced both IgG and IgM. Finally, expansion of peripheral B cells from tetanus toxoid-immunized donors with either HMW-BCGF or LMW-BCGF revealed that the HMW-BCGF-expanded population produced predominantly IgG tetanus-specific antibody in the presence of BCDF (in the absence of HMW-BCGF), whereas the LMW-BCGF-expanded population produced IgM much greater than IgG tetanus-specific antibody. Thus, HMW-BCGF may function to expand a subpopulation of B cells for memory B cell functions.

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. IV. Immune reconstitution.

Studies of postmyeloablative immune reconstitution have been reported for allogeneic bone marrow transplantation and also for non-T cell-depleted autologous/syngeneic BMT. However, there is a paucity of information regarding immune recovery following T cell-depleted autologous/syngeneic BMT. We have developed a primate transplantation tolerance model in which rhesus monkeys were conditioned with total-body irradiation and extensively T cell-depleted autologous BMT and given a major histocompatibility complex-mismatched heterotopic cardiac allograft. This model provided an opportunity to study peripheral immune recovery following T cell-depleted autologous BMT. Limiting dilution analysis was used to quantify marrow T cells following depletion (2.8% to 25.6% marrow T cells predepletion, 0.00014% to 0.036% residual marrow T cells postdepletion). We found that (1) hematopoietic engraftment was prompt despite extensive marrow T cell depletion, (2) reconstitution of CD4+ helper T cells and CD8+ cytotoxic T cells were substantially delayed (6-12 months) compared with the recovery of CD8+ suppressor T cells, CD16+ NK cells, and CD20+ B cells, (3) distinction between CD8+ cytotoxic T cells and CD8+ suppressor T cells by the CD28 marker was critical in revealing the markedly discrepant recoveries of those subsets, and (4) immune reconstitution resembled that observed in recipients of T cell-depleted allogeneic and non-T cell-depleted autologous/syngeneic BMT, suggesting that the pattern of immune recovery following BMT is not substantially influenced by either allogeneic effects or the number of transferred T cells over a range of values.

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants.

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.

Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-gamma production.

Studies of cell-surface molecules involved in human T cell interaction reveal that differential expression of each of three adhesion molecules (LFA-3, CD2, and LFA-1) subdivides human peripheral blood T cells into major subpopulations. Systematic analysis of the relationship between expression of these and other markers of T cell subsets demonstrates a single major subset of human peripheral blood T lymphocytes distinguished by enhanced expression of LFA-3, CD2, LFA-1, and three other markers (CDw29 [4B4], UCHL1, and Pgp-1). Large differences in relative expression are observed for UCHL1 (29-fold) and LFA-3 (greater than 8-fold), and smaller differences (2- to 4-fold) are seen for CDw29, CD2, LFA-1, and Pgp-1. Bimodal distribution of LFA-3 is found on both CD4+ cells and on CD8+ cells as well as on B lymphocytes (CD19+). Neonatal T cells (CD3+) are comprised almost exclusively of the subset expressing low LFA-3, CD2, LFA-1, CDw29, and UCHL1. Activation of cord peripheral blood mononuclear leukocytes with PHA leads to uniform enhanced expression of each of these molecules on CD3+ cells. Functional analyses of these T cell subsets were performed after sorting of adult T cells based on differential LFA-3 expression. Only the LFA-3+ subset proliferated in response to the Ag tetanus toxoid, even though the LFA-3- subset proliferated more strongly to PHA. Furthermore, the LFA-3+ subset made greater than fivefold more IFN-gamma than the LFA-3- subset in response to PHA, despite the fact that both subsets made equivalent amounts of IL-2. This phenotypic and functional analysis of resting and activated newborn and adult T cells indicates that human memory T cells express enhanced levels of LFA-3, CD2, LFA-1, UCHL1, CDw29, and Pgp-1; we speculate that the increase in expression of T cell adhesion molecules LFA-3, CD2, and LFA-1 on memory cells is functionally important in their enhanced responsiveness.

Enhanced antigen-presenting capacity of cultured Langerhans' cells is associated with markedly increased expression of Ia antigen.

Recent studies indicate that when epidermal Langerhans' cells (LC) are cultured for 2 to 3 days they, in comparison to freshly prepared LC, exhibit markedly enhanced ability to stimulate T cell proliferative responses in oxidative mitogenesis and in the mixed epidermal-leukocyte reaction. In this study, we determined whether cultured LC enhance antigen-specific T cell responses, and whether such enhanced stimulatory capacity correlates with the level of Ia antigen expressed on LC. We used C3H/He (Iak) epidermal cells as stimulators and, as responder cells, both the trinitrophenyl-specific clones D8 and SE4, which were assayed for [3H]dThd incorporation, and the pigeon cytochrome c specific hybridoma 2C2, which was assayed for interleukin 2 production. Cultured LC induced 10 to 100 times greater proliferation or interleukin 2 production by responder cells than did freshly prepared LC. The intensity of I-Ak and I-Ek, expressed on cultured LC as assessed by immunofluorescence and flow cytometry, was found to be 10 to 36 times greater on a per cell basis than that on freshly prepared LC. Depletion of LC from fresh epidermal cell suspensions by anti-Iak and complement or treatment with 50 mJ/cm2 medium range ultraviolet light or cycloheximide before culture abrogated both the increase in Ia expression and antigen-specific clonal proliferation. The results suggest that when LC are removed from their usual epidermal milieu, they express increased amounts of Ia and become more potent stimulators of T cell responses.

Abnormal distribution of epididymal antigens on spermatozoa from infertile men.

An antiserum raised against human epididymal proteins associated with ejaculated sperm was used to test the hypothesis that the amount and/or localization of these antigens may be altered in men with infertility. With the use of immunofluorescence we found that in sperm from fertile donors 88.4% of the cells had the antigens localized over the acrosomal cap only and 1.3% had most of the antigens at extraacrosomal sites. Fifteen of the 26 infertile men (P1) studied had a similar relative distribution of antigens, but the remaining 11 patients (P2) had a 38-fold increase in cells with extraacrosomal localization of the antigens (40%, P less than 0.005). Using flow cytometry to quantitate immunofluorescence, content of antigen on sperm from patients from population P1 (680 +/- 60 V X 10(-4)) was not different from that of control (835 +/- 53 V X 10(-4], whereas it was significantly lower in sperm from patients from population P2 (554 +/- 64 V X 10(-4), P less than 0.005). Differences could not be correlated with parameters measured by routine semen analysis. Our results suggest a possible relationship between the decreased amount of epididymal antigens or their altered localization on sperm and the infertility of patients from population P2.

Generation of activated killer cells in tumor-bearing hosts.

Activated killer (AK) cells were generated in spleen-cell cultures derived from tumor-bearing hosts (TS) whereas, under the same conditions, cultured normal spleen cells (NS) gave little cytotoxicity. The AK effectors were primarily Thy1+, AGM1- and Lyt2- and thus were neither classic cytotoxic T lymphocytes (CTL) nor classic NK cells. These AK cells selectively killed tumor targets of different etiologic origins and did not kill concanavalin-A-induced lymphoblasts. The broad target-cell reactivity of these AK cells was also confirmed by cold target-inhibition experiments. Generation of AK cell correlated with interleukin-2 (IL-2) production, and the levels of AK cells generation paralleled those of IL-2 production. Furthermore, the generation of AK cells was blocked by the anti-IL-2 receptor monoclonal antibody (MAb) (alpha IL-2R), indicating that IL-2 was involved, and thus these AK cells were lymphokine-activated killer (LAK) cells. We previously showed that the expression of AGM1 on LAK precursors disappeared when they differentiated into LAK effectors, indicating that the activated LAK cells lacked AGM1. When examining the serologic phenotype of the LAK precursors in tumor-bearing hosts, we found that they lacked AGM1, which suggested that these LAK precursors were in an "activated" state. These cells were still Thy1-, and were thus different from fully activated LAK effectors which were Thy1+ cells, indicating that the full differentiation of LAK cells in vivo was arrested in the tumor-bearing hosts. We also found that the presence of small amounts of X-irradiated tumor cells prevented the generation of AK cells. These findings suggest that, in the tumor-bearing hosts, the presence of tumor cells triggers the activation of AK precursors; however, the same tumor cells may also be immunosuppressive, which prevents the full differentiation of AK precursors into AK effectors.

Ia+ murine epidermal Langerhans cells are deficient in surface expression of the class I major histocompatibility complex.

Murine epidermal Langerhans cells were analyzed with fluorescence microscopy and multicolor flow cytometry for the surface expression of major histocompatibility complex (MHC) class I and class II antigens. Langerhans cells of H-2k haplotype were identified in situ or in epidermal-cell suspensions by their surface expression of the MHC class II determinants I-Ak and I-Ek. More than 90% of class II-positive Langerhans cells in epidermal-cell suspensions expressed no or barely detectable amounts of MHC class I antigens. Quantitation by flow cytometry revealed that H-2k Langerhans cells expressed only 1.6-3.3% as much H-2Kk as did class II-negative keratinocytes in the same epidermal-cell suspensions. By fluorescence microscopy, class I MHC antigens were not detectable on Langerhans cells in situ when analyzed on sheets of intact epidermis. The deficient expression of class I MHC permitted highly purified Langerhans cell populations to be isolated from epidermal cell suspensions by treatment with anti-class I MHC monoclonal antibody and complement. It is likely that the uniquely low cell-surface expression of class I MHC antigen by Langerhans cells has relevance to both immune responses in the skin as well as to mechanisms of skin allograft rejection. In addition, it is conceivable that regulation of class I MHC expression on antigen-presenting cells in general is an important but hitherto unrecognized mechanism of immune regulation.

Culture and characterization of murine dendritic Thy-1+ epidermal cells.

Although numerous advances have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of the murine Thy-1+ dendritic epidermal cell (Thy-1+ EC), elucidation of its functional qualities has been hampered by the difficulty in preparing pure populations of these cells. We therefore sought to obtain expanded, purified populations of Thy-1+ EC using culture techniques. Since Thy-1+ EC are bone marrow-derived, density gradient enriched populations of freshly harvested epidermal cells (FH-EC) were placed in culture under conditions known or suspected to promote mitogenesis among leukocyte subsets. FH-EC prepared from truncal skin of C3H/HeN mice (Thy-1.2+) were cultured at 37 degrees C in 5% CO2 in complete medium (CM) of Eagle's Hanks' amino acid with 10% fetal calf serum, nutrients, and antibiotics at 10(6) FH-EC/well in 24-well culture plates. CM was supplemented with one or more of the following: concanavalin A (Con-A), interleukin-1/epidermal cell-derived thymocyte-activating factor (IL-1/ETAF), IL-2, IL-3, gamma interferon, indomethacin (IM), and anti-Thy-1.2 antibody. Media with appropriate supplements were changed every 2-3 days. Freshly isolated, enriched FH-EC contained 7-20% Thy-1+ EC (defined as brightly fluorescing cells readily distinguishable from weakly fluorescing keratinocytes), which also stained with antibodies directed against asialo GM1, Ly 5.1, and vimentin but did not stain with antibodies to other T cell-, B cell- or macrophage phenotypic markers. Analysis of 10 separate cultures revealed a 3- to 10-fold expansion of nonkeratinocyte Thy-1+ cells after 21 +/- 4 days in culture in CM supplemented with Con-A and IM, and 70-100% of viable cells after expansion were Thy-1+. Phenotypic analysis of expanded cells revealed the emergence in 10 separate cultures of one of two mutually exclusive distinct populations: one Thy-1+, asialo GM1+, L3T4- (natural killer phenotype) and the other Thy-1+, asialo GM1-, L3T4+ (T helper phenotype). Experiments designed to explain the emergence of an L3T4+ population suggest that phenotypic modulation occurred in vitro.