Class II major histocompatibility complex-restricted T cells specific for a virion structural protein that do not recognize exogenous influenza virus. Evidence that presentation of labile T cell determinants is favored by endogenous antigen synthesis.

The contribution of viral infectivity to the expression of MHC class II-restricted T cell determinants was studied. A murine I-Ed-restricted T cell hybridoma recognizing the neuraminidase (NA) glycoprotein of influenza PR8 virus was stimulated strongly by infectious virus but failed to recognize antigen introduced on noninfectious virions. Recognition correlated with the de novo synthesis of viral NA within infected APC. The effectiveness of infectious virus did not depend strictly upon the amount of NA present in cultures, since high NA concentrations could be achieved by addition of nonreplicative virus without being stimulatory for NA-specific T cells. Recognition of a determinant generated only when synthesized in murine host cells was ruled out, since, in high concentration, NA isolated from purified egg-grown virions, even if reduced and alkylated, was recognized by the T hybridoma clone. Isolated NA was recognized when added to pre-fixed APC, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. Data suggest that endogenously synthesized antigen may contribute most significantly to presentation of labile T cell determinants. In addition to NA, recognition of an I-Ed-restricted determinant of the influenza hemagglutinin (HA) molecule, shown previously to have a relatively short half-life on APC surfaces, was enhanced greatly by infectious virus. In contrast, T cell recognition of a more stably expressed I-Ed-restricted site of the same HA polypeptide was only marginally improved on infected APC.

Quantitation of influenza virus antigens on infected target cells and their recognition by cross-reactive cytotoxic T cells.

Monoclonal antibody to type-A influenza virus matrix (M)-protein was used to quantitate the appearance of M-protein on abortively infected P815 cells. After 16 h of infection with different type-A viruses, only a low amount of M-protein appears on the surface of infected cells (approximately 10(3) site/cell) in contrast to approximately 10(5) hemagglutinin molecules on each cell surface. However, virus replication is required for M-protein appearance. Analysis of solubilized membranes purified from 16-h-infected cells shows approximately 10(4) M-protein molecule/cell in the plasma membrane, a content that is consistent with the observed low surface expression, and that indicates that most of the M-protein is localized internally. We found no evidence that cross-reactive cytotoxic T cells could recognize M-protein; neither monoclonal antibody or hyperimmune anti-M-protein antiserum could inhibit T cell killing, either alone or in combination with monoclonal anti-H-2 antibody. Taken together, the low level of M-protein appearance and lack of T cell blocking by anti-M-protein antibody leaves doubt that M-protein is the antigen recognized by cross-reactive cytotoxic T cells.

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

Virus entry and antigen biosynthesis in the processing and presentation of class-II MHC-restricted T-cell determinants of influenza virus.

Receptor-mediated uptake of influenza virus is responsible for efficient introduction of virus particles to APC. This leads to the effective presentation to T-cells of very small concentrations of proteins entering on the intact virus. Endocytosed virus transits rapidly to the endosome compartment. Entry into this environment appears to greatly affect the fate of T-cell determinants. While promoting the presentation of determinants which require extensive antigen processing, the intracellular environment appears also to lead to destruction of labile determinants, such as those of NA. The same NA determinants are efficiently presented by actively infected cells, indicating that newly biosynthesized viral proteins need not be subjected to the same handling as internalized viral particles. In a similar way, site 3 of HA, which, in a single pulse of noninfectious virus or isolated HA protein is expressed with a relatively short half-life, has greatly improved levels of duration and expression on actively infected APC. Since certain T(H) determinants are unavailable or poorly expressed when introduced on nonreplicative influenza virus, vaccination with inactivated virus might have limitations in stimulating T(H) as well as class-I responses. Finally, individual T-cell determinants of the same protein can exhibit distinct patterns of expression and persistence on APC surfaces. These different half-lives of T(H) determinants may be influential in determining immuno-dominance of T-cell sites. Determinants that are longer-lived on APC may have a greater probability of interacting with appropriate T(H) precursors, which could lead to an enhanced T-cell response to that region of the viral protein.

On the epidemiology of yaws in African miners (1942).

Several points of interest arise from these unusual human yaws infections: (i) European miners contracted a treponemal infection from Africans with yaws and developed yaws; (ii) the infectious patients who started the minor epidemics in the outbreaks must have been relapses from prolonged latent early infections; (iii) the time taken for the treponemes, possibly only a few minutes, to penetrate the skin through a minor injury seemed to be less than six hours; (iv) transmission occurred only at the working rock face deep underground with a tropical climate, and not on the surface 1,630 metres above sea-level, where there were abundant opportunities; (v) the search for treponemes in the environment of the working place was restricted by the inability to culture the treponeme.

McLetchie on mass campaigns.

PIP: Dr. J.L. McLetchie was asked in 1963 to express his thoughts on the many aspects of mass campaigns for the historical record fro future field workers. The significance of his thoughts at that time lies in the soundness of the principles outlined, based upon field responsibility. It was from such principles that the modern strategy of community health in dveloping countries arose, which was adopted and put into practice by the World Health Organization and was presented at the Alma Ata Conference on Primary Health Care in 1978. The text is reproduced here. There should be no need to argue the need for mass campaigns under conditions as they exist at present in Africa as well as other tropical areas. Several conditions cannot be dealt with in other way, e.g., tuberculosis, malnutrition, onchocerciasis, yaws, sleeping sickness. The most essential needs are the recognition, at the highest political and administrative level, that a country's services must be balanced, with well-developed preventive, laboratory, and curative sections. To obtain and retain this balance requires strong and continous administrative action to counteract the overwhelming attraction of the curative services to young African doctors and to expatriates on short-term contracts. The preventive services divide naturally into those dealing with urban problems having a large content of environmental hygiene and those dealing with rural problems in which curative medicine plays a mojor part, i.e., mass treatment. In rural health work, the "amateur" -- the young medical officer assigned to rural duties for a period of 1-2 years -- may play a valuable part but cannot do so unless the service is well organized and has a core of "professionals," senior medical staff with considerable experience with rural problems and how to tackle them. Rural health specialists have to work closely in cooperation with other sections of the medical department, with other departments, and with local government authorities. The position of the local government authority is important to mass campaigns and rural health work generally. Mass campaignd are usually initiated by special teams of mobile staff which progress from district to district, often leaving behind in each district 1 or more members to begin the consolidation phases. At an early stage in a country's development there may be 1 mass campaign directed against 1 specific disease. The initial campaign and the follow-up or consolidation may then be relatively simple. Staff are trained in a single simple technique or in a few diagnostic and therapeutic techniques. Each local authority should eventually have a sufficiency of multipurpose staff to meet the area's expected needs in the final consolidation phase.^ieng

An introduction to diagnostic criteria of syphilis, treponarid and yaws (treponematoses) in dry bones, and some implications.

Diagnostic criteria of syphilis and some other diseases are proposed from a study of 424 crania and calvariae and 250 long bones in 22 medical museums in Europe. Yaws bone lesions in Uganda and changes in Australian aboriginal bones also contributed to the establishment of these criteria. Any deductions about disease in the past or isolated populations must depend upon acceptable diagnostic criteria; post mortem damage must be recognised. In crania and calvariae the sequence of changes of Virchow's caries sicca, and in long bones nodes/expansions with superficial cavitation are sound indicators of syphilis, and of yaws and treponarid in relevant geographical areas. Attention is called to the cause of sequestra in European calvariae labelled syphilis, the absence of sequestra due to haematogenous pyogenic osteomyelitis in Australian and other aboriginal bones and possibly in Europe before the Middle Ages. The number of bones with diagnostic criteria needed to demonstrate the endemicity of a particular infection in a past community is discussed. There is also need for an extensive application of diagnostic criteria of syphilis to pre-Columbian or pre-European bones everywhere. The uncertain future of old dry diseased bones in medical museums, and the need for reference centres to provide sound advice and guidance in palaeopathology are stressed.

Class I H-2d-restricted cytotoxic T lymphocytes recognize the neuraminidase glycoprotein of influenza virus subtype N1.

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) that recognize the neuraminidase (NA) glycoprotein of subtype N1 influenza A viruses have been demonstrated in BALB/c mice. Responses to NA were obtained only in protocols that use two in vivo inoculations of virus, including a recombinant vaccinia virus containing the NA of subtype N1 influenza virus (NA-VAC) to prime or boost. Restimulation in vitro was also required for CTL recognition of NA and strongly depended on the specific N1 virus used. Influenza viruses A/Puerto Rico/8/34 (H1N1), A/CAM/46 (H1N1), J1 (H3N1), and JAP/BEL (H2N1), but not A/Bellamy (H1N1) or MEM/BEL (H3N1) virus, were able to stimulate NA-specific memory T cells in vitro. Single or double in vivo inoculation of any of the N1 viruses or a single injection of NA-VAC failed to elicit restimulatable NA-specific CTL. Lysis of NA-VAC-infected cells at low effector/target ratios was comparable to that observed toward other influenza virus proteins known to be major targets of CTL in BALB/c mice, indicating that antigenic determinants of the subtype N1 NA molecule can be efficiently presented in the context of major histocompatibility complex class I.

Activated T hybridomas induce upregulation of B7-1 on bystander B lymphoma cells by a contact-dependent interaction utilizing CD40 ligand.

The upregulation of costimulatory molecules of antigen presenting cells (APC) resulting from interaction with activated T cells was studied in an in vitro system composed of well-characterized murine T hybridomas and B cell lymphomas. Increased B7-1 expression was induced on both MHC-matched and -mismatched (bystander) B lymphoma cells present in cultures of activated T hybridomas. Identical results were obtained with T hybridomas activated by either the appropriate peptide presented by MHC-matched APC or by mitogen stimulation in the absence of MHC/TCR cognate interactions. Soluble factors alone did not lead to upregulation of B7-1; B lymphomas cultured on the opposite side of a transwell membrane from an ongoing T cell stimulation response, or in supernatants of activated T cells, did not exhibit enhanced expression of B7-1. Antibodies to the CD40 ligand (CD40L) of T cells inhibited the increased appearance of B7-1 on B lymphomas. Significant B7-1 upregulation on the population of bystander B cells could be achieved even when they were present at a 10:1 excess over MHC-matched APC. These data indicate that B7-1 upregulation results from contact between bystander B cells and activated T hybridomas in vitro by CD40-CD40L interaction, without the requirement for TCR/MHC interaction.

Immunologic tolerance for immune system-mediated diseases.

Induction of long-term, antigen-specific immunologic unresponsiveness holds great promise for the treatment of many immune system-mediated diseases, including asthma, allergies, autoimmune diseases, and transplant rejection. Unlike current immunosuppressive treatments, immunologic tolerance therapies would affect only the undesired immune responses, leaving protective immunity intact. A variety of approaches to immunologic tolerance induction are being taken, reflecting the molecular and cellular complexity of immune system activation and regulation. The presentations summarized in this report represent promising strategies, some of which are being evaluated in advanced animal models and human clinical trials. Approaches presented include the following: interference with costimulatory signals in T-cell induction, T-cell receptor antagonism by altered peptides, exploitation of antigen-induced apoptosis to eliminate undesired T cells, opposition of inflammation by the induction of regulatory cytokines, induction of transplant tolerance by mixed chimerism, and deviation from deleterious allergic antibody responses by use of immunostimulatory DNA sequences. These multifaceted approaches are strongly supported by knowledge of basic immune mechanisms, which should facilitate the rational development of these therapies for controlling immune-mediated diseases.

Ultrastructure of developing ascospores in Sordaria brevicollis.

The ultrastructure of ascospore wall formation in the pyrenomycete Sordaria brevicollis was studied in developing asci at progressive time intervals. From early spore delimitation through final stage of maturation, the wall of the ascospore differentiated into four composite layers, the periascosporium the delineation ascosporium, the subascosproium, and the endoascosproium, While ascospores were at the hyaline stage of development,they possessed only the periascosporium and delineation ascosporium as their wall components. At about 7 to 8 days from the initiation of the cross, the spores developed a yellow color, and this coloration was always associated with the elaboration of the subascorsporium just internal to the ascosporium. Asthe spores continued to progressively darken in color, the subascosporium was seen to increase in complexity, electron density, and thickness. Soon after the formation of the subascosporium, the endoascosporium began to develop de novo and was, therefore, the last wall layer formed as the spore approached maturity.

Influenza-specific suppression: contribution of major viral proteins to the generation and function of T suppressor cells.

Suppressor cells were generated in BALB/c mice by two sequential injections of PR8 influenza virus (A/Puerto Rico/8/34[H1N1]) and were tested for their ability to inhibit proliferative cellular responses towards multiple viral and nonviral antigens. In this way, suppression specific to PR8 as compared with purified protein derivative (PPD) and keyhole limpet hemocyanin (KLH) antigenic responses was illustrated. Experiments involving adoptive transfer of suppression to naive hosts with subfractionated lymphocyte populations demonstrated that the suppressors were Lyt-2+ T cells. Two major questions were addressed with this system. First, a determination was made of which anti-viral protein proliferative responses were affected by the PR8-induced T suppressor (Ts) cells. Ts cells were found to inhibit proliferating cells with specificities for isolated hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix (M) antigens. Second, experiments were conducted to analyze the viral proteins contributing to the induction of PR8-specific Ts cells. Inoculations with either isolated HA or a combination of M + NP proteins induced T suppression specific to proliferative responses towards PR8. These experiments illustrate the contribution of external (HA and NA) as well as internal (M + NP) viral proteins to Ts cell generation and function.

A convenient culture chamber for observation and embedding of macrophage monolayers for transmission electron microscopy.

A chamber adapted from a polypropylene test tube is described for the cultivation and processing of undisturbed monolayers of normal mouse macrophages for transmission electron microscopy (TEM). Peritoneal exudate cells are grown in the chambers on Visking dialysis membranes pretreated with Polybrene (Sigma Chemical Co.) and normal mouse serum. The cells are not further disturbed after adherence since the entire chamber, including dialysis membrane and cells, is processed for electron microscopy using standard material and protocols and the embedded dialysis membrane is readily oriented and thin-sectioned. This method is efficient for studying adherent cells since growth can be nearly confluent and cells remain firmly attached throughout the manipulations. Since a transparent membrane is used, cells can also be observed with the light microscope at all stages prior to sectioning.

H-2 expression by lymphoid cells of different mouse strains: quantitative interaction of H-2 with monoclonal antibodies and their Fab fragments.

Monoclonal antibodies (H100-30/3 and 11-4.1) to H-2k were used to study H-2 antigen expression and characteristics of the H-2 antigen-antibody interaction at the cell surface. Studies with radiolabelled F(ab')2 and Fab' fragments of 11-4.1 antibody confirmed that monoclonal IgG binding to cells is directly proportional to the number of H-2 sites and shows a high proportion of monovalent binding over a wide range of concentrations. Scatchard plots showed no difference in the binding affinity constant (Ka) of a given monoclonal antibody on lymphoblasts from various H-2k F1 and congenic strains, but only in the number of antigenic sites per cell. F1 (k x d) lymphoblasts show 1 x 10(5) H-2k sites/cell, about 50% of the expression in homozygotes. Dk expression in C3H.OH is 1.4 x 10(4) sites/cell. While normal cells appear to have a constant amount of H-2 (2-3 x 10(5) sites/cell), BW thymoma cells show unstable H-2 expression, having an average of five times fewer H-2 sites per cell when grown in vitro as compared to in vivo growth. Another BW cell surface marker, Thy-1.1, does not fluctuate in parallel with H-2. The 30/3 and 11-4.1 antibodies bind to topologically distinct sites on H-2Kk. The binding of these antibodies can be perturbed differentially: paraformaldehyde fixation of cells abolishes binding of 11-4.1 antibody but not of 30/3 antibody; increasing temperature increases the Ka of 30/3 antibody binding but decreases the Ka of 11-4.1 antibody binding to cells.

H-2 and viral haemagglutinin expression by influenza-infected cells; the proteins are close but do not cocap.

The proximity of H-2K and D antigens and influenza virus haemagglutinin (HA) molecules on the surface of infected target cells was assessed by a topographical study using monoclonal antibodies to H-2 and to HA. The effect of pretreatment of fixed, infected cells with excess of one monoclonal antibody on the subsequent binding of a second radiolabelled antibody was measured. Using CBA mouse B lymphoblasts which were paraformaldehyde fixed 5 hr postinfection with influenza virus (A/USSR/90/77), pretreatment with monoclonal antibody 30/3 to H-2Kk and Dk partially blocked (Approximately equal to 37%) the binding of one radiolabelled monoclonal anti-HA antibody (264/2). A different monoclonal IgG (W18/1) directed to the same HA molecule was not blocked by similar pretreatment of cells with the anti-H-2 antibody. Interaction of monoclonal antibodies with their sites is highly specific, and mutual blocking of two antibodies requires very closely located sites even if the antibodies are directed to the same molecule. We therefore have evidence for proximity of H-2 and HA molecules; however, we were unable to demonstrate cocapping of H-2K and D antigens with influenza HA.