Human monoclonal antibodies against Pseudomonas aeruginosa lipopolysaccharide derived from transgenic mice containing megabase human immunoglobulin loci are opsonic and protective against fatal pseudomonas sepsis.

Pseudomonas aeruginosa is a significant human pathogen, and no vaccine is commercially available. Passive antibody prophylaxis using monoclonal antibodies (MAb) against protective P. aeruginosa epitopes is an alternative strategy for preventing P. aeruginosa infection, but mouse MAb are not suitable for use in humans. Polyclonal human antibodies from multiple donors have variable antibody titers, and human MAb are difficult to make. We used immunoglobulin-inactivated transgenic mice reconstituted with megabase-size human immunoglobulin loci to generate a human MAb against the polysaccharide (PS) portion of the lipopolysaccharide O side chain of a common pathogenic serogroup of P. aeruginosa, 06ad. The anti-PS human immunoglobulin G2 MAb made from mice immunized with heat-killed P. aeruginosa was specific for serogroup 06ad pseudomonas. The MAb was highly opsonic for the uptake and killing of P. aeruginosa by human polymorphonuclear leukocytes in the presence of human complement. In addition, 25 microg of the MAb protected 100% of neutropenic mice from fatal P. aeruginosa sepsis. DNA sequence analysis of the genes encoding the MAb revealed V(H)3 and Vkappa2/A2 variable-region genes, similar to variable-region genes in humans immunized with bacterial PS and associated with high-avidity anti-PS antibodies. We conclude that human MAb to P. aeruginosa made in these transgenic mice are highly protective and that these mice mimic the antibody response seen in humans immunized with T-cell-independent antigens such as bacterial PS.

Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies.

The major impediment to the development of murine monoclonal antibodies (mAbs) for therapy in humans has been the difficulty in reducing their potential immunogenicity. XenoMouse¿trade mark omitted¿ mice obviate this problem while retaining the relative ease of generating mAbs from a mouse. XenoMouse strains include germline-configured, megabase-sized YACs carrying portions of the human IgH and Igkappa loci, including the majority of the variable region repertoire, the genes for Cmicro, Cdelta and either Cgamma1, Cgamma2, or Cgamma4, as well as the cis elements required for their function. The IgH and Igkappa transgenes were bred onto a genetic background deficient in production of murine immunoglobulin. The large and complex human variable region repertoire encoded on the Ig transgenes in XenoMouse strains support the development of large peripheral B cell compartments and the generation of a diverse primary immune repertoire similar to that from adult humans. Immunization of XenoMouse mice with human antigens routinely results in a robust secondary immune response, which can ultimately be captured as a large panel of antigen-specific fully human IgGkappa mAbs of sub-nanomolar affinities. Monoclonal antibodies from XenoMouse animals have been shown to have therapeutic potential both in vitro and in vivo, and appear to have the pharmacokinetics of normal human antibodies based on human clinical trials. The utility of XenoMouse strains for the generation of large panels of high-affinity, fully human mAbs can be made available to researchers in the academic and private sectors, and should accelerate the development and application of mAbs as therapeutics for human disease.

Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes.

The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and kappa light (L) chain loci. Each of the two H and the two kappa chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique VH (5 versus 66) or Vkappa genes (3 versus 32). Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by VH and Vkappa gene complexity: (a) production of functional mu chains at the transition from the pre B-I to the pre B-II stage; (b) productive VkappaJkappa recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of mu chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.

Stories of spiritual awakening. The nature of spirituality in recovery.

Substance abuse has had a devastating impact on the lives of millions. As substance use and abuse continues to ravage communities, researchers remain in the dark about what works to ensure successful recovery from addiction. In searching for the answers, researchers have often overlooked the role of religious and spiritual practices and beliefs in preventing use and relapse. The study reported here describes the process of spiritual awakenings experienced by some persons in recovery during their quest for sobriety. The data suggests that persons in recovery often undergo life altering transformations as a result of embracing a power higher than one's self, that is, a Higher Power. The result is often an intense spiritual journey that leads to sustained abstinence. Given how widespread substance abuse is, research on the nature, implications, and limitations of a spiritual approach to addiction might offer new options for treatment.

Injury and anomie: effects of violence on an inner-city community.

OBJECTIVES: Widespread violence affects individuals but also alters group life. This study was designed to examine the effects of violence on an inner-city community. METHODS: A qualitative study was undertaken that included field observations and semistructured interviews. The study took place in Washington Heights, a New York City neighborhood with a high rate of violence, largely secondary to the drug trade. RESULTS: The 100 people interviewed differed widely in their definitions of violence and in their likelihood of having experienced violent acts in the course of daily life. High, medium, and low violence microenvironments were identified; risk of exposure to violence, but not individual definitions of violence, differed by location. Violence in all parts of the neighborhood inhibited social interactions, but the intensity of this effect differed by microenvironment. CONCLUSIONS: In Washington Heights, violence has injured individuals and fractured social relationships, leading to the state of social disarray referred to as "anomie." The public health response to the violence epidemic should address anomie through community organizing efforts.

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Production of antigen-specific human antibodies from mice engineered with human heavy and light chain YACs.

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells.

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

Germ-line transmission and expression of a human-derived yeast artificial chromosome.

Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.

Two types of genetic interaction implicate the whirligig gene of Drosophila melanogaster in microtubule organization in the flagellar axoneme.

The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an alpha-tubulin locus, alpha 1t, mutations in a beta-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known alpha- or beta-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrlnc4 and B2tn are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2tn, for a deficiency of alpha 1t, or for the haync2 allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrlnc4 or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.

Correlation between tubulin mRNA stability and poly(A) length over the cell cycle of Physarum polycephalum.

During the cell cycle of the Physarum polycephalum plasmodium, levels of alpha-tubulin mRNA rise exponentially in G2 phase, reach a peak at metaphase 40-fold above basal levels, and then fall exponentially to basal levels after mitosis. We show that post-mitotic alpha-tubulin mRNA carries poly(A) tracts of less than 30 residues. By contrast, when levels of alpha-tubulin mRNA rise during G2 phase, the mRNA has a poly(A) tract of approximately 80 bases. The length of the poly(A) tract of any mRNA encoding actin is relatively constant at fewer than 30 bases through the cycle. We have estimated the apparent rate of synthesis of alpha-tubulin mRNA at different stages of the cell cycle by short-term labeling in vivo. Transcription of alpha-tubulin mRNA continues even after mitosis, though the rate may be diminished relative to that in late G2 phase. So, the post-mitotic molecular half-life of alpha-tubulin mRNA must be less than the 19 minute half-life by which the levels of this species fall. The fact that the apparent rate of alpha-tubulin mRNA synthesis is not vastly greater in early G2 phase than in post-mitotic plasmodia is consistent with an S-phase destabilization of alpha-tubulin mRNA molecules. Thus, the poly(A) tail is shorter when the alpha-tubulin mRNA is less stable.

Developmental regulation and identification of an isotype encoded by altB, an alpha-tubulin locus in Physarum polycephalum.

A subcloned portion of the 5' nontranslated sequence from a Physarum alpha-tubulin cDNA is specific for a single alpha-tubulin locus, altB, of Physarum polycephalum. We find that this locus is expressed only in the plasmodium and encodes at least an alpha 1-tubulin isotype, which we have designated alpha 1B. Hybridization patterns of other subclones of this cDNA reveal two sequences for alpha-tubulin at the altB locus.

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

Value orientation and psychosocial adjustment at various levels of marijuana use.

Personality characteristics and values of 475 undergraduates were studied with respect to marijuana use and the usual demographic variables. Frequent Users and Adamant Nonusers (i.e., those persons who "never have used marijuana and never will") each account for 23% of the sample. The demographic data were essentially similar between the four groups under study, but the group differences in reported marijuana use corresponded to large differences in personality and values. The latter was assessed with an instrument developed by the authors based on F. Kluckhohn's theory of variation in value orientations; personality characteristics were assessed with the California Psychological Inventory. The latter (CPI) portrays the Frequent User, in comparison to the other groups but especially relative to the Adamant Nonuser, as likely to be interpersonally and intellectually more effective, trusting in others, and confident and to possess a greater degree of ego strength. The Adamant Nonuser appears to be more submissive in general, lacking in self-insight, dependent on external structure, and judgmental. These differences are interpreted in terms of the value orientations that have primacy for the members of each of the groups. For example, the Adamant Nonuser feels that nature is more subjugating than do the other groups. His standards are extrinsic, and he has a desire for order, associated with an emphasis on authority supported by a belief that humankind is naturally evil or dangerous and that one.