Herb species inclusion in grazing swards for dairy cows-A systematic review and meta-analysis.

A systematic review and meta-analysis were conducted to estimate the effect of herb species on milk production and urinary nitrogen (UN) excretion from grazing dairy cows. Grazing swards consisting of herb species grown with either a grass species or a grass and legume (multispecies swards) were compared with non-herb-containing swards consisting of a grass species grown as a monoculture or grass-legume swards (simple swards). A literature search was completed using the online databases CAB Direct, Web of Science, and Google Scholar, using the search strategy "dairy cow", "herb OR forb OR phorb", and "grazing". Milk production data, variance, and sample size were required for eligibility. In all, 116 studies were identified. Following eligibility screening, 11 papers from 6 journals, published between 2006 and 2018, were available for analysis. Studies were from New Zealand (N = 7), Australia (N = 3), and the United States (N = 1). The population was either Holstein Friesian or Holstein Friesian × Jersey dairy cows, with a range in mean daily milk yield (MY) from 12.1 kg to 34.7 kg (mean = 18.6 kg). A total of 25 comparisons were used for milk production analysis, with 324 and 284 cows included in multispecies and simple sward groups respectively. Data analysis was conducted in R using a random effects, robust variance estimation model (R Foundation for Statistical Computing, Vienna, Austria). Heterogeneity was reported using the I2 statistic. Milk production was significantly increased. Analysis of MY resulted in a weighted mean difference (WMD) of +1.20 kg/d (95% CI = 0.90, 1.49; I2 = 4%). Fat and protein kg were also significantly increased (WMD +0.06 kg/d; CI = 0.01, 0.11). Urinary nitrogen excretion was estimated from milk urea nitrogen when reported (n = 6). A WMD of -28.1 g of N/d (95% CI = -81.1, 24.9) was generated, with heterogeneity high among studies (I2 = 75%). This meta-analysis shows the potential benefits of multispecies swards. Although we saw no significant difference in UN excretion, an increase in milk production was found.

Lessons learnt conducting minimally invasive autopsies in private mortuaries as part of HIV and tuberculosis research in South Africa.

Current estimates of the burden of tuberculosis (TB) disease and cause-specific mortality in human immunodeficiency virus (HIV) positive people rely heavily on indirect methods that are less reliable for ascertaining individual-level causes of death and on mathematical models. Minimally invasive autopsy (MIA) is useful for diagnosing infectious diseases, provides a reasonable proxy for the gold standard in cause of death ascertainment (complete diagnostic autopsy) and, used routinely, could improve cause-specific mortality estimates. From our experience in performing MIAs in HIV-positive adults in private mortuaries in South Africa (during the Lesedi Kamoso Study), we describe the challenges we faced and make recommendations for the conduct of MIA in future studies or surveillance programmes, including strategies for effective communication, approaches to obtaining informed consent, risk management for staff and efficient preparation for the procedure.

Les estimations actuelles du poids de la tuberculose (TB) maladie et de la mortalité qui lui est due parmi les patients positifs à l'infection par le virus de l'immunodéficience humaine (VIH) dépendent beaucoup de méthodes indirectes, qui sont moins fiables pour vérifier les causes de décès au niveau individuel et de modèles mathématiques. Une autopsie peu invasive (MIA) est utile au diagnostic de maladies infectieuses, fournit une approximation raisonnable de l'étalon or de la vérification de la cause du décès c'est-à-dire une autopsie diagnostique complète. Si elle est utilisée en routine, elle pourrait améliorer les estimations de mortalité spécifique d'une cause. A partir de nos expériences de MIA sur des adultes positifs au VIH dans des morgues privées d'Afrique du Sud (au cours de l'étude Lesedi Kamoso), nous décrivons les défis rencontrés et faisons des recommandations pour la réalisation de MIA dans des études futures ou des programmes de surveillance, incluant des stratégies de communication efficaces, des approches visant à obtenir un consentement éclairé, une prise en charge du risque pour le personnel et une préparation efficace de la procédure.

Las estimaciones actuales de morbilidad por tuberculosis (TB) y de mortalidad por causas específicas en las personas positivas frente al virus de la inmunodeficiencia humana (VIH) se fundamentan en su mayor parte en métodos indirectos que son menos fiables para determinar las causas de muerte individuales y en modelizaciones matemáticas. La autopsia mínimamente invasiva (MIA) es útil en el diagnóstico de las enfermedades infecciosas, ofrece un sustituto aceptable al método de referencia para determinar la causa de muerte (que es la autopsia diagnóstica completa), y cuando se usa de manera sistemática, mejora las estimaciones de la mortalidad por causas específicas. A partir de su experiencia con la MIA en adultos con infección por el VIH en empresas fúnebres privadas en Suráfrica (durante el estudio Lesedi Kamoso), los autores describen las dificultades que encontraron y formulan recomendaciones que se pueden aplicar en el futuro al realizar la autopsia mínimamente invasiva en estudios de investigación o en programas de vigilancia; se preconizan estrategias de comunicación efectivas, métodos de obtención del consentimiento informado, la gestión de riesgos para el personal y la preparación eficiente del procedimiento.

Implementation and Operational Research: What Happens After a Negative Test for Tuberculosis? Evaluating Adherence to TB Diagnostic Algorithms in South African Primary Health Clinics.

INTRODUCTION AND BACKGROUND: Diagnostic tests for tuberculosis (TB) using sputum have suboptimal sensitivity among HIV-positive persons. We assessed health care worker adherence to TB diagnostic algorithms after negative sputum test results. METHODS: The XTEND (Xpert for TB-Evaluating a New Diagnostic) trial compared outcomes among people tested for TB in primary care clinics using Xpert MTB/RIF vs. smear microscopy as the initial test. We analyzed data from XTEND participants who were HIV positive or HIV status unknown, whose initial sputum Xpert MTB/RIF or microscopy result was negative. If chest radiography, sputum culture, or hospital referral took place, the algorithm for TB diagnosis was considered followed. Analysis of intervention (Xpert MTB/RIF) effect on algorithm adherence used methods for cluster-randomized trials with small number of clusters. RESULTS: Among 4037 XTEND participants with initial negative test results, 2155 (53%) reported being or testing HIV positive and 540 (14%) had unknown HIV status. Among 2155 HIV-positive participants [684 (32%) male, mean age 37 years (range, 18-79 years)], there was evidence of algorithm adherence among 515 (24%). Adherence was less likely among persons tested initially with Xpert MTB/RIF vs. smear [14% (142/1031) vs. 32% (364/1122), adjusted risk ratio 0.34 (95% CI: 0.17 to 0.65)] and for participants with unknown vs. positive HIV status [59/540 (11%) vs. 507/2155 (24%)]. CONCLUSIONS: We observed poorer adherence to TB diagnostic algorithms among HIV-positive persons tested initially with Xpert MTB/RIF vs. microscopy. Poor adherence to TB diagnostic algorithms and incomplete coverage of HIV testing represents a missed opportunity to diagnose TB and HIV, and may contribute to TB mortality.

High incidence of latent tuberculous infection among South African health workers: an urgent call for action.

SETTING: In South Africa, health care workers (HCWs) are at two-fold greater risk of acquiring tuberculosis (TB) disease than the general population. Few studies have evaluated the risk of incident tuberculous infection. OBJECTIVE: To determine the incidence and risk factors for latent tuberculous infection (LTBI) among HCWs and to compare the results of the interferon-gamma release assay (IGRA) with those of the tuberculin skin test (TST). DESIGN: HCWs, including medical students, underwent a TST and human immunodeficiency virus (HIV) and IGRA testing at baseline and 12 months, and IGRA at 6 months. The participants kept 12-month TB exposure logs. RESULTS: Among 199 participants (150 [76%] females, median age 31 years [range 20-61]), incident LTBI was documented using IGRA in 25/97 (26%; incident rate 29 cases/100 person-years [py], 95%CI 20-44) and using TST in 25/93 (27%; incident rate 29 cases/100 py, 95%CI 19-42). Agreement between TST and IGRA was poor (44.8%, κ = 0.23). Higher annual exposure to TB cases was reported among persons with LTBI than in those who were persistently IGRA-negative (81 cases, 95%CI 61-102 vs. 50 cases, 95%CI 43-57, P < 0.01). CONCLUSION: The high LTBI incidence and the association of incident LTBI with annual TB caseload among HCWs indicate that more effective TB infection control should be implemented in South African health care facilities.

Effects of mutations in potential phosphorylation sites on transcytosis of FcRn.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across intestinal epithelial cells of suckling rats and mice from the lumenal surface to the serosal surface. In cell culture models FcRn transports IgG bidirectionally, but there are differences in the mechanisms of transport in the two directions. We investigated the effects of mutations in the cytoplasmic domain of FcRn on apical to basolateral and basolateral to apical transport of Fc across rat inner medullary collecting duct (IMCD) cells. Basolateral to apical transport did not depend upon determinants in the cytoplasmic domain. In contrast, an essentially tailless FcRn was markedly impaired in apical to basolateral transport. Using truncation and substitution mutants, we identified serine-313 and serine-319 as phosphorylation sites in the cytoplasmic domain of FcRn expressed in Rat1 fibroblasts. Mutations at Ser-319 did not affect transcytosis across IMCD cells. FcRn-S313A was impaired in apical to basolateral transcytosis to the same extent as tailless FcRn, whereas FcRn-S313D transported at wild-type levels. FcRn-S313A recycled more Fc to the apical medium than the wild-type receptor, suggesting that Ser-313 is required to allow FcRn to be diverted from an apical recycling pathway to a transcytotic pathway.

Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells.

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Control of an outbreak of vancomycin-resistant Enterococcus faecium in an oncology ward in South Africa: effective use of limited resources.

An outbreak of vancomycin-resistant enterococci (VRE) occurred in an adult oncology ward of a large teaching hospital in Johannesburg, South Africa. The outbreak strain was identified as an Enterococcus faecium carrying the vanA resistance genotype. Macro-restriction analysis showed that the majority of strains were clonally related. Modified infection control interventions were implemented and control of the outbreak was achieved. Although the epidemiology of VRE is well documented in Europe, North America and Australia, this problem has only recently emerged in South Africa. The epidemiology of the outbreak appears similar to that described for outbreaks elsewhere.

Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across cellular barriers between mother and offspring. FcRn also protects circulating IgG from catabolism, probably during transport across the capillary endothelium. Only one cell culture model of transcytosis has been used extensively, the transport of IgA from the basolateral to the apical surface of Madin-Darby canine kidney cells by the polymeric immunoglobulin receptor (pIgR). We report that rat inner medullary collecting duct (IMCD) cells transfected with DNA encoding the (alpha) subunit of rat FcRn specifically and saturably transport Fc when grown as polarized monolayers. Using this system, we have found that transcytosis by FcRn, like transcytosis by the pIgR, depends upon an intact microtubule system. FcRn differs most strikingly from the pIgR in its ability to transport its ligand in both the apical to basolateral and basolateral to apical directions. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibited basolateral to apical transport by FcRn more than apical to basolateral transport, suggesting that there are differences in the mechanisms of transport in the two directions. Lastly, we found that transcytosis by FcRn depends upon vesicular acidification. We anticipate that the IMCD cell culture model will allow further elucidation of the mechanism of IgG transport by FcRn.

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Basolateral but not apical application of protease results in a rapid rise of transepithelial electrical resistance and formation of aberrant tight junction strands in MDCK cells.

In the presence of Ca2+, application of trypsin to the basolateral surface of confluent MDCK cell monolayers with formed tight junctions (TJ), induces the formation of basolaterally oriented aberrant TJ strands. Induction of aberrant TJ strands is accompanied by an increase in transepithelial electrical resistance (TER), up to 90%, which upon addition of trypsin inhibitor is maintained for up to 1 h. Thereafter TER returns slowly to baseline values. Under similar conditions, application of trypsin to the apical surface has little or no effect on either TER or the number of aberrant TJ strands. Confocal microscopy of monolayers, immunostained for ZO-1, revealed that this TJ associated cytoplasmic protein, extended below the TJ along the basolateral surface following brief exposure to trypsin. Removing Ca2+ after treatment of the monolayer with basolaterally applied trypsin resulted, after 20 min, in the increased partitioning of TJ particles onto the E fracture face, of both normal and aberrant TJ strands. Like the TJ strands themselves, therefore, aberrant strands may be linked to cytoskeletal elements. Aberrant TJ strands do not form when monolayers, maintained in low Ca2+ medium, are exposed to trypsin, suggesting that under these conditions TJ precursors, and/or trypsin-sensitive proteins regulating TJ strand assembly, are sequestered in a vesicular compartment that is inaccessible to exogenous trypsin. Prolonged exposure of the apical surface of an established, polarized epithelium with intact TJ to trypsin, had little effect on TJ integrity and did not induce aberrant strands.

Interstitial lung macrophages interact with dendritic cells to present antigenic peptides derived from particulate antigens to T cells.

When the protective structural and functional barriers of the lung are breached, immune responses must be generated in order to contain invading micro-organisms. This requires the presence of accessory cells capable of phagocytosing and presenting immunogenic peptides to either naive or sensitized T cells. In contrast to dendritic cells (DC) present in the airway epithelium, those within the lung parenchyma do not readily engulf particulates and, therefore, other mechanisms must account for their apparent ability to present immunogenic peptides derived from micro-organisms. The purpose of the present study was to determine the extent to which interstitial macrophages (IM) interact with lung DC to process and present antigenic peptides, derived from particulate, heat-killed Listeria monocytogenes (HKL), to HKL-immune T cells. Results show that highly purified Ia- lung IM avidly phagocytose fluorescent-labelled HKL, but they do not present antigen to primed T cells. Their ability to present antigen is only modestly increased following interferon-gamma (IFN-gamma) stimulation. Conversely, mature DC isolated from the lung interstitium do not phagocytose fluorescent-labelled HKL. In antigen presentation assays, however, addition of 10% (2.5 x 10(3)/ml) Ia- IM to DC and HKL results in a two- to threefold increase in antigen presentation by DC to HKL-immune T cells. Conditioned medium (CM), generated by 2.5 x 10(4)/ml IM induced to phagocytose HKL, when administered to DC and HKL-sensitized T cells without added intact HKL, resulted in brisk mitogenesis, a response that did not occur in T cells sensitized to an irrelevant antigen. Conditioned medium derived from larger numbers of IM was inhibitory. When IM phagocytosed inert polystyrene beads, the resulting CM induced modest T-cell mitogenesis, suggesting that small amounts of cytokines were released. The results indicate that in small numbers, IM augment DC function, in part, by the release of antigenic peptides which are then presented by DC to T cells. When present in numbers greater than 50% of DC, however, they inhibit DC function, probably due to the release of soluble inhibitors.

Ontogeny of Ia+ accessory cells in fetal and newborn rat lung.

In the adult mammalian lung, Ia+ dendritic cells (DC) constitute a significant population of immunologically potent accessory cells that are important in the regulation of immune responses to inhaled antigens. The newborn, in most species, displays an increased susceptibility to sensitization by inhaled antigens; whether an immaturity of pulmonary accessory cells is involved has not been determined. In the present study, the ontogeny and function of these cells were examined in fetal and newborn rats. Cells identified as DC in fetal and newborn rat lungs were Ia+, C11b+/-, OX41-, OX43-, W3/13-, W3/25-, and OX8-. They were characterized ultrastructurally by an eccentric, lobulated nucleus, a paucity of lysosomes, delicate cytoplasmic processes, and abundant membrane-associated Ia. Ia+ DC were first detected within the pulmonary mesenchyme at day 15 and by day 17 of gestation they were also present within the epithelium lining airways. The appearance of Ia+ DC preceded the migration of either T4 or T8 subclasses of T cells to the lung, the latter becoming significant only after birth, when the newborn was exposed to environmental antigens. In none of the fetal or newborn animals was Ia detected on alveolar type II cells. The accessory cell function of rat pulmonary DC, isolated from fetuses at 20 and 21 days of gestation and from newborns, was tested by an autologous mixed leukocyte reaction. At 20 and 21 days of gestation, pulmonary DC were 40 and 60% as effective, respectively, in stimulating cell proliferation in purified autologous adult splenic T cells as those isolated from adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection.

Dendritic cells (DC), in general, and pulmonary DC, in particular, are a heterogeneous population of cells, their phenotype and function being dependent on their anatomic location, their state of activation, and the regulatory effect of locally secreted cytokines. Using a novel microdissection technique, the epithelium from the trachea and entire airway system was harvested, and the contained DC isolated at greater than 90% purity. The phenotype and function of these airway DC (ADC) was compared to DC isolated, at greater than 90% purity, from the parenchyma of the same lung. In contrast to lung DC (LDC), ADC did not express intercellular adhesion molecule 1 (ICAM-1) in situ, the amount of immune associated antigen (Ia) expressed was less (as determined by immunoperoxidase staining and immunopanning), and greater than 50% of ADC displayed Fc receptors (FcR). The majority of LDC were ICAM-1+, less than 5% expressed FcR, and all were intensely Ia+. Airway DC were most numerous in tracheal epithelium, but they were also present in small numbers in the epithelium of the most distal airways. Their numbers increased in all segments of the tracheobronchial epithelium in response to the administration of IFN-gamma. ADC were consistently more effective than LDC in presenting soluble (hen egg lysozyme) and particulate (heat-killed Listeria monocytogenes) antigens to antigen-sensitized T cells. By contrast, LDC were significantly more efficient in stimulating the proliferation of nonsensitized T cells in an autologous mixed leukocyte reaction. These data suggest that in normal animals, intraepithelial DC of airways share many attributes with Langerhans cells of the skin. Interstitial LDC, by contrast, reside in an environment where they may be exposed to a different set of regulatory factors and where they have progressed to a more advanced stage of differentiation than ADC. Both groups of DC are, however, heterogeneous, reflecting the continuous turnover that these cells undergo in the lung.

Accessory cells of the lung. I. Interferon-gamma increases Ia+ dendritic cells in the lung without augmenting their accessory activities.

Dendritic cells are specifically adapted to provide accessory signals for the growth of T lymphocytes. Ia+ dendritic cells are present within the normal lung; however, little is known concerning their regulation in vivo. Interferon-gamma (IFN-gamma) is a proinflammatory lymphokine that augments the expression of Ia antigens and promotes the accessory activities of a variety of cells. In order to determine whether IFN-gamma regulates pulmonary dendritic cells in vivo, Lewis rats were injected intraperitoneally with recombinant murine IFN-gamma (2 x 10(5) U/rat/day) or with buffered saline for 5 consecutive days. Following sacrifice, the lungs were excised, and the distribution and number of Ia (OX-6)+ cells was determined in situ. Dendritic cells were localized in the mucosal lining of the tracheobronchial tree, in pulmonary capillaries, as well as in the alveolar septal interstitium and subjacent to the pleural surfaces. IFN-gamma yielded a specific increase in Ia+ dendritic cells in alveolar septa and in pulmonary airways. Purified Ia+ dendritic cells from enzymatic digests of lung were excellent accessory cells for the proliferative responses of both antigen-primed and naive T lymphocytes. IFN-gamma did not, however, further augment the expression of Ia antigens or the accessory activities of pulmonary dendritic cells. These results suggest that IFN-gamma may promote pulmonary T cell-mediated inflammatory responses in vivo by increasing the number of Ia+ dendritic accessory cells in the lung.

Antigen-independent binding of T-cells by dendritic cells and alveolar macrophages in the rat.

The antigen-independent binding of CD4+ T-lymphoblasts by alveolar and peritoneal macrophages and splenic dendritic cells (DC) was compared. DC formed clusters with T-lymphoblasts within 30 min at 37 degrees C, whereas alveolar and peritoneal macrophages did not. Antigen-independent binding developed between macrophages and CD4+ blasts by 4 h at 37 degrees C. Binding by alveolar macrophages was trypsin sensitive, magnesium dependent, serum independent, and cold insensitive, whereas binding by DC required serum and was inhibited by cold. Cluster formation (cell aggregates greater than 250 microns 2) by macrophages and CD4+ blasts was increased by interferon-gamma and phorbol esters, but diminished by lipopolysaccharide. However, each of these factors increased cluster formation by blasts with DC. Efforts to promote antigen-independent binding of T cells by Ia+ macrophages did not alter their poor accessory cell capacities. The role of cluster formation in accessory cell activities was examined. Inhibitors of DC clustering, including trypsin, paraformaldehyde, and tunicamycin, abrogated the ability of DC to support antigen presentation and lectin-mediated proliferation. It is concluded that rapid antigen-independent binding to T-cells is a distinct property that is restricted to DC. Exposure to LPS may down regulate nonproductive binding of T-cells to alveolar macrophages. Our data further suggest that accessory cell activities in the rat are not a function of alveolar macrophages and may be limited to specialized Ia+ cells of dendritic lineage.

The poor accessory cell function of macrophages in the rat may reflect their inability to form clusters with T cells.

The accessory cell functions of Ia+ alveolar and peritoneal macrophages were compared to those of splenic cells in the rat. Whereas splenic mononuclear cells and dendritic cells were excellent supporters of both MHC-restricted and nonrestricted T-cell mitogenic responses, Ia+ macrophages were inefficient antigen-presenting cells and poor supporters of lectin mitogenic responses. Binding of antigen-primed T-cell blasts by splenic cells in the presence of Con A or antigen occurred within 30 min and subsequently led to the formation of nonadherent clusters of "dendritic-like cells" and proliferating T-cell blasts. Unstimulated Ia- macrophages failed to bind T cells during 30 min of coculture but formed conjugates with T-cell blasts within 24 hr. Delayed binding did not require the presence of antigen or lectin, or the expression of Ia antigens by the macrophage, and did not lead to T-cell proliferation. Antigen-specific binding and antigen presentation, but not lectin mitogenesis, were enhanced by treating antigen-pulsed Ia+ macrophages with neuraminidase for 30 min at 37 degrees C. Neuraminidase did not augment splenic accessory cell function. Antigen-specific binding of T cells to Ia+ macrophages and accessory cell function may be enhanced by desialation of glycoproteins on the cell surface membrane.

Flow-cytometric and ultrastructural analysis of alveolar macrophage maturation.

Alveolar macrophages (AM) from adult and newborn rats were studied by flow cytometry and ultrastructural morphometry. We observed that the laser scatter and autofluorescent properties of newborn macrophages were different from those of adult cells. Relative to the adult AM, the forward-angle laser scatter obtained with the newborn AM was reduced; this optical measurement appeared to correlate with the smaller mean size, as determined by ultrastructural and electronic volume measurements. The diminished right-angle laser scatter (90 degrees angle) correlated with the presence of fewer small, irregularly shaped lysosomal structures in the newborn AM, compared with AM from adult animals. AM from 1-2-day-old rats displayed large vacuoles containing multilamellar structures, which proved to be less effective at scattering light. Cells from newborn rats were less autofluorescent, a finding that appeared to correlate best with the numbers of secondary lysosomes. Flow cytometry may be used to discern structural alterations that occur during the maturation of AM. These changes correlate well with quantitative ultrastructural analyses of these cells.