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Infective endocarditis (IE) is associated with significant morbidity and mortality. Its diagnosis can be especially challenging, as can the identification of a causative pathogen, which, in turn, is crucial for appropriate management. Here, we present a rare case of Streptococcus intermedius endocarditis complicated by lung and cerebral abscess in which clinicians employed cell-free deoxyribonucleic acid (cfDNA) testing to confirm the diagnosis, establish a causative pathogen, and determine appropriate antibiotic therapy. Notably, the positive cfDNA test prevented the need for brain biopsy in this case and has implications for diagnostic and therapeutic guidelines., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Hennis et al.)

Retroviral constructs were designed to express the novel cytokine interleukin 18 (IL-18), also known as interferon-gamma-inducing factor, in a murine neuroblastoma cell line [neuro-2a (N-2a)] to examine the effects of IL-18 expression on tumorigenicity. N-2a cells expressing proIL-18 (N-2a/IL-18p) were as tumorigenic as parental N-2a cells, whereas N-2a cells engineered to secrete mature IL-18 (N-2a/IL-18m) were nontumorigenic. Inoculation of mice with N-2a/IL-18m generated immediate immunity to parental N-2a. N-2a/IL-18m formed tumors in mice depleted of CD4+ and CD8+ T cells, suggesting that the antitumor immune response was T cell mediated. The resulting T-helper (Th) immune response was also characterized in vitro and had a large Th1 component based on in vitro production of the cytokines IFN-gamma and granulocyte macrophage colony-stimulating factor in response to tumor cells and IL-18.

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.

Efficient retroviral gene transfer into hematopoietic stem and progenitor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragments. In this paper, we further optimize this technology for human CD34+ cells. Investigating the role of cytokine prestimulation in retrovirus-mediated gene transfer on plates coated with the recombinant FN CH-296 revealed that prestimulation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) CD34+ cells was essential to achieve efficient gene transfer into clonogenic cells. The highest gene transfer occurred by prestimulating PB CD34+ cells for 40 hr with a combination of stem cell factor (SCF), G-CSF, and megakaryocyte growth and development factor (MGDF) prior to retroviral infection on CH-296. Surprisingly, a prolonged simultaneous exposure of primary CD34+ PB cells to retrovirus and cytokines in the presence of CH-296 lowered the gene transfer efficiency. Gene transfer into cytokine prestimulated CD34+ bone marrow (BM) cells was not influenced by increasing the coating concentrations of a recombinant FN fragment, CH-296, nor was it adversely influenced by increasing the number of CD34+ target cells, suggesting that the amount of retroviral particles present in the supernatant was not a limiting factor for transduction of CD34+ BM cells on CH-296-coated plates. The polycation Polybrene was not required for efficient transduction of hematopoietic cells in the presence of CH-296. Furthermore, we demonstrated that repeated exposure of CH-296 to retrovirus containing supernatant, called preloading, can be employed to concentrate the amount of retroviral particles bound to CH-296. These findings establish a simple and short clinically applicable transduction protocol that targets up to 68% of BM or G-CSF-mobilized PB CD34+ cells and is capable of genetically modifying up to 17% of CD34+CD38-/dim PB cells.

The T cell co-stimulatory molecule B7-1 was transduced into a poorly immunogenic murine neuroblastoma cell line (Neuro-2a, N-2a) alone or in combination with MHC class II genes to test the ability of these genes to stimulate antitumor immunity. N-2a cells transduced with B7-1 exhibited reduced tumorigenicity, whereas N-2a cells overexpressing both MHC class II (syngeneic, I-Ak) and B7-1 totally abrogated tumorigenicity. Rejection of I-Ak/B7-1 cells was dependent on both CD4+ and CD8+ T cells. The ability of both vaccines to induce protection against parental N-2a was temporally dependent on the time of secondary N-2a challenge. To investigate the immunity generated by N-2a/B7-1 and N-2a/I-Ak/B7-1 vaccines, we tested the ability of these modified cells to stimulate in vitro the proliferation of syngeneic splenocytes from naive mice. A significant increase in splenocyte proliferation was observed with N-2a/I-Ak/B7-1 cells compared to N-2a cells. We also determined that vaccination with N-2a/I-Ak/B7-1 cells was able to generate cytotoxic T cell responses to unmodified N-2a cells. The introduction of B7-1 and I-Ak into N-2a was able to convert a poorly immunogenic tumor to a highly immunogenic one; however, mice bearing large established unmodified tumors had little response to vaccination with N-2a/I-Ak/B7-1 cells. Our results emphasize the importance of tumor immunogenicity in the treatment of established tumors with MHC class II/B7-1 tumor cell vaccines.

Malignant tumors express tumor-related antigens, but effective antitumor immunity does not occur in the primary host. One hypothesis is that there is insufficient stimulation of T-cell responses due to ineffective antigen presentation. An approach to overcome these deficiencies is to modify tumor cells to express major histocompatibility complex (MHC) class II genes and thus facilitate the presentation of antigens directly by tumor cells. Our experiments with a murine neuroblastoma cell line (neuro-2a) transduced with DR (xenogeneic), 1-Ab (allogeneic), or 1-Ak (syngeneic) MHC class II genes support this notion. The relative potencies of the modified neuro-2a to induce immunity to unmodified neuro-2a were neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak. Modified neuro-2a also could stimulate naive splenocyte proliferation in vitro. The relative magnitude of the proliferative responses seen after stimulation with modified tumor cells was neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak > unmodified neuro-2a. Hence, the tumor cell-induced splenocyte proliferative responses observed in vitro correlate with the effectiveness of the tumor cell vaccines to induce antitumor immunity in vivo. These data show that the expression of exogenous MHC class II on tumor cells is a potent stimulus for specific antitumor immunity. Because of the correlation of the in vivo and in vitro immune responses to modified tumor cells, the tumor-induced lymphocyte proliferation assay may be useful in evaluating tumor cell vaccines produced by additional genetic modifications of tumor cells.

Immunological recognition of transformed cells is critically important to limit tumor development and proliferation. Because established tumors have escaped immune recognition and elimination, novel strategies to enhance antitumor immunity have been developed. A unique approach has used the introduction of genes encoding major histocompatibility complex (MHC) antigens into tumor cells. Experiments in mice have shown that the expression of syngeneic class II MHC antigens in tumor cells completely abrogates tumorigenicity and induces tumor-specific immunity. In this study we sought to determine whether a more effective antitumor immune response would be generated by introducing xenogeneic class II MHC genes into tumor cells. To address this question we used recombinant retroviruses to express human class II MHC genes in a highly malignant murine neuroblastoma cell line, Neuro-2a. We found that normal mice inoculated with Neuro-2a expressing the human class II MHC antigen did not develop tumors and were immune to subsequent challenge with unmodified Neuro-2a cells. In addition, mice bearing small established Neuro-2a tumors were cured by vaccination with Neuro-2a expressing human class II MHC. We hypothesize that a similar approach using retroviral-mediated transduction of class II MHC genes into human tumor cells may be an effective alternative to current cancer treatment.

Three retroviral vectors, containing a human adenosine deaminase (ADA) cDNA linked to either the simian virus 40 (SV40) early promoter, the human cytomegalovirus (CMV) immediate early promoter, or the Moloney murine leukemia virus (MoMLV) promoter, were tested for their ability to express ADA following infection and transplantation of murine bone marrow. Virus was produced by using PA317 amphotropic retrovirus packaging cells. The titer of each of the vectors was similar and no helper virus was detected. Human ADA was expressed in the blood of some animals for 6 months after transplantation of infected marrow, and vector DNA was found in the spleen and in bone marrow from these animals. The percentage of animals expressing human ADA (33%) and the amount of human ADA in blood (1-5% of total ADA) was similar for each of the vectors. These results show that amphotropic vectors are capable of infecting pluripotent hematopoietic stem cells having long-term repopulating ability, and that a variety of promoters allow gene expression following differentiation of these early cells.

The binding of mouse epidermal growth factor-urogastrone (EGF-URO) to membranes from term human placenta is peptide-specific, saturable (about 20 pmol of EGF-URO bound maximally/mg of protein), reversible, and of high affinity (KD about 400 pM). Optimal binding is observed at pH 7.6. At low pH (3.5 to 5.0). EGF-URO can be reversibly dissociated from the receptor; however, exposure to pH < 3 irreversibly inactivates the receptor. The binding, which does not exhibit ligand cooperativity, exhibits an association rate constant of 6.1 x 10(-4) s-1 and a dissociation rate constant of 6.1 x 10(-4) s-1. The dissociation constant determined from the rate constants, 240 pM, is in reasonable agreement with the constant estimated by equilibrium methods. Both monovalent and divalent cations augment EGF-URO binding 2- to 3-fold. Although in general, divalent cations enhance binding at lower concentrations (optimum, 5 mM) than do monovalent cations (optimum, approximately 80 mM), there is no cation-specific effect. Neither guanine nor adenine nucleotides affect EGF-URO binding. Whereas the proteolytic enzymes (trypsin, chymotrypsin, papain, and pepsin) inactivate the receptor, neuraminidase and phospholipases A2, C, and D augment EGF-URO binding. Neuraminidase increases the number of available sites without affecting ligand affinity. Wheat germ agglutinin, concanavalin A, and phytohemagglutinin all compete for the binding of EGF-URO. The data complement previous observations of EGF-URO binding obtained in intact cells and provide a basis for the solubilization, characterization, and isolation of this receptor from a rich tissue source.

The purpose of this study was systematically to evaluate the effectiveness of several modes of psychological intervention used with male asthmatic children being treated in the Allergy Outpatient Clinic. Therapeutic effectiveness was measured by large airway changes in respiratory function, and the number of recurrent asthmatic attacks. The psychotherapeutic modes used were Relaxation Training, Assertive Training, and combined Relaxation plus Assertive Training. All patients were administered medication by the responsible physician. The group psychotherapy experiences were controlled by using patients who received medication alone and by patients who received medication and met in a leaderless group. The effectiveness of the therapeutic interventions was determined by comparisons between pretreatment measures and measurements taken during and after the eight-week treatment program. Both Relaxation Training by itself and combined Relaxation plus Assertive Training increased respiratory functioning and reduced the number of attacks. Assertive Training alone failed to improve respiratory function and had a tendency to increase the frequency of asthmatic attacks. It was concluded that the most effective management in male asthmatic children was achieved by the combination of medical and psychological treatments.

Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with (125)I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with (125)I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization.

The historical view of masculinity/femininity posited essentially bipolar opposites, with the presence of one set of characteristics precluding the other. More recent studies of sex-role stereotypes have defined sexual orientation within clusters of socially desirable attributes which males and females perceive as differentiating males from females. This view negates the contention that psychological sex roles are composed of bipolar opposites, and concludes that the constructs of masculinity and femininity are independent dimensions rather than a single bipolar dimension. Little is known about the sex-role functioning of adolescents, yet it is during adolescence that qualitative shifts occur in interpersonal relationships and concurrent changes occur in cognitive functioning, with adolescents shifting toward hypothetical thinking and abstract ideal notions. In view of these changes, much can be learned about adult functioning by studying the sex-role perceptions of adolescents related to familial and social variables. This study examines the sex-role perceptions that adolescents hold of fathers, mothers, ideal males, ideal females, and selves. Differences exist between male and female adolescents, and significant linkages exist between sex-role identification and academic achievement.

Hemophilia B is an X-chromosome-linked bleeding disorder resulting from lack of clotting factor IX activity and affects about 1 in 30,000 males. Current therapy involves injection of crude factor IX prepared from pooled human plasma. Treatment is complicated by viral contaminants in factor IX preparations, such as non A-non B hepatitis and the AIDS virus, and by the practical difficulties of chronic injections. An alternative therapy might include the insertion of a factor IX expression vector into the somatic cells of affected individuals to allow continued production of factor IX. Toward this end, we have constructed a retrovirus vector for transfer and expression of factor IX. Despite the fact that factor IX is normally synthesized in hepatocytes and requires extensive post-translational modification for activity, we have shown that fully active factor IX can be made by human skin-derived fibroblasts. These results open the way to testing the use of skin grafts for gene therapy of hemophilia B.

We describe the solubilization and purification of both the photoaffinity-labeled and unlabeled human placenta receptor for epidermal growth factor-urogastrone (EGF-URO). The photolabeled receptor can be purified 300- to 500-fold from membrane extracts by combined immunoaffinity and lectin-agarose affinity chromatography. This isolation approaches theoretical purity. Upon gel filtration and wheat germ agglutinin-Sepharose chromatography, the photolabeled receptor and the EGF-URO binding activity co-migrate, as measured by a newly developed lectin-agarose immobilization assay of receptor binding. Sequential ion exchange, Cibacron blue-Sepharose, wheat germ agglutinin-Sepharose, and gel filtration chromatography yield a 110-fold purification of the EGF-URO binding activity; this purified fraction contains protein constituents that exhibit electrophoretic mobilities parallel to those of the photolabeled receptor constituents, that have apparent molecular weights of 180,000 and 160,000. This molecular weight range is consistent with a measured apparent Stokes radius for both the photolabeled and unlabeled receptor of 5.1 nm (Sephacryl S-200 gel filtration), although an apparent radius of 4.3 nm is estimated by gel filtration on Sepharose-6B. The apparent molecular weight of the photolabeled receptor is unaffected by 2-mercaptoethanol. This work provides a basis for further detailed studies of this growth factor receptor.

The objective of management in the pregnant diabetic patient is to achieve physiologic glucose homeostasis through the use of diet and insulin. As outlined, the numerous ancillary tests developed during the past 15 years to assist the clinician in determining impending fetal death have left much to be desired, especially where metabolic homeostasis has not been achieved prior to the thirty-sixth week of gestation. The statistics from this institution indicate that the maintenance of the plasma glucose concentration below 100 mg. per cent throughout gestation, regardless of the severity of the diabetes, all but removes the risk of maternal-fetal complications due to diabetes. The management is uniform for all patients exhibiting an abnormality of carbohydrate metabolism, and, although it is rather difficult to accept, there have been minimal neonatal complications when the protocol outlined in this presentation has been followed.

Patients with certain genetic disorders can be cured by bone marrow transplantation. However, as prospective donors do not exist for most patients with potentially curable genetic abnormalities, an alternative treatment for such patients involves the transfer of cloned genes into the patient's haematopoietic stem cells followed by re-infusion of the treated cells. Retroviral vectors provide an efficient means for transferring genes into mammalian cells and have been used to transfer genes into mouse haematopoietic cells. We have now produced amphotropic retroviral vectors containing either the bacterial gene for neomycin resistance or a mutant dihydrofolate reductase gene that confers resistance to methotrexate and have used these vectors to infect and confer drug resistance to human haematopoietic progenitor cells in vitro. Transfer could be demonstrated in the absence of helper virus by using an amphotropic retrovirus packaging cell line, PA12 (ref. 9). These studies are an important step towards the eventual application of retrovirus-mediated gene transfer to human gene therapy and for molecular approaches to the study of human haematopoiesis.

Retroviral vectors with an internal cytomegalovirus major immediate-early gene enhancer/promoter regulating HLA class II gene expression were used to transfer HLA cDNA into human EBV-transformed B-lymphoblastoid cell lines. HLA-DQ2 beta and DQ3.2 beta cDNA were transferred into DQ3.2 and DQ2 homozygous lymphoblastoid cell lines, respectively. Serologic analysis of the infected cell lines with allospecific mAb demonstrated surface expression of these exogenous DQ molecules implying that DQ alpha-chains from DR3, DQ2-positive cells can pair with DQ3.2 beta-chains and, similarly, DQ alpha-chains from DR4, DQ3.2-positive cells can pair with DQ2 beta-chains. Immunoprecipitation of the introduced DQ3.2 beta molecule resulted in co-purification of the allotype-mismatched endogenous DQ2 alpha polypeptide. We also show that vectors with a cytomegalovirus major immediate-early gene enhancer/promoter result in higher levels of expression of the transduced gene compared to previously described HLA vectors with either the SV-40 early enhancer/promoter or the lymphotropic papovavirus-enhanced SV-40 promoter. Although deletion of HLA cDNA did sometimes occur in the process of generating virus-producing clones, the HLA cDNA is stably maintained in virus-producing clones, once it is generated. This retroviral expression system is a highly efficient way to study HLA gene function.

The evaluation of a behavior and development training program that was integrated into a general pediatric training residency is reported. Thirty-two residents who participated in the program were evaluated over one year on measures designed to assess residents' opinions about the relevance of behavior and development concepts, residents' perceptions of their own competence in behavior and development knowledge and skills, supervisors' ratings of residents' general clinical performance, and residents' attitudes about 10 specific illnesses. Results suggest that residents perceive a high relevance for psychosocial concepts and an increasing competence with these concepts after training. Establishing rapport with patients emerged as an important variable in supervisors' ratings of resident performance and in residents' self-ratings of competence. Attitudes were initially negative toward the more behavioral and psychophysiological disease entities but showed some positive change over the year. These results and their implications are discussed.

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.