An improved method to assess the encapsulation response in arthropods.

Ecoimmunology explores how ecological factors and evolutionary processes influence immune responses across various taxa and how immune responses trade-off with other traits. Studying immune responses requires biologically meaningful immunoassays applicable to a broad range of taxa and are sensitive enough to detect changes in the immune response. Useful immunoassays should also correlate with immunocompetence and fitness. The encapsulation response, a complex immune mechanism in arthropods, serves as a robust method for ecoimmunological investigations. However, traditional methods to test the encapsulation response can require long training. This study introduces an innovative, cost-effective method for assessing the encapsulation immune response in arthropods, which simplifies the procedure by reducing the training time and skill required. Our modified device utilizes a pen and syringe assembly for inserting monofilaments into arthropod larvae. We compared our device against traditional methods. Despite the new method being 22% faster, it did not compromise the accuracy or effectiveness of the encapsulation response when compared with traditional techniques, demonstrating similar degrees of melanization and encapsulation. Our method allowed for more accessible participation by less experienced researchers, such as undergraduates, facilitating their involvement in ecoimmunological research.

Identification of Covalent Cyclic Peptide Inhibitors in mRNA Display.

Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein-protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.

Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4.

MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.

Beyond intimacy: conceptualizing sex differences in same-sex friendships.

This study was designed to replicate and extend prior findings that same-sex friendships of women and men are equally important but that women's friendships are more intimate. A group of adolescents and a group of adults were asked to complete an anonymous questionnaire regarding the quality of their close friendships, the degree to which they would support a close friend in times of difficulty, and the degree to which they would celebrate with a friend in times of success. Results demonstrated that both females and males saw and spoke with their close friends and were equally willing to confront and trust their close friends. Females, however, reported more desire to spend time with a close friend in times of difficulty and to celebrate with a close friend who had just experienced a positive event. Results are discussed in terms of the differing functions of women's and men's same-sex friendships and women's greater general interest in and attention to transitions in the lives of other individuals.

Prevalence of male pattern hair loss in 18-49 year old men.

BACKGROUND: Previous studies investigating the prevalence of male pattern hair loss (MPHL) typically used biased samples of men recruited from clinical populations which may limit generalizability of findings to broader populations. OBJECTIVE: To obtain an updated and improved estimate of the occurrence of MPHL in healthy men residing in the community. METHODS: Community-based sample of healthy men aged 18-49 years participated in a study investigating the effects of MPHL. Participants completed a brief questionnaire self reporting degree of hair loss, general health-related quality of life (HRQL) and hair-loss-specific measures. A trained observer also rated each participant using standardized classification for MPHL. RESULTS: The proportion of men with moderate to extensive hair loss (type III or greater) was 42%. The proportion of men with moderate to extensive hair loss increased with increasing age, ranging from 16% for men 18-29 years of age to 53% of men 40-49. Twelve percent of the men were classified as having predominantly frontal baldness (type A variants). CONCLUSIONS: MPHL, especially frontal baldness, may be more common than previously reported.

Effects of self-perceived hair loss in a community sample of men.

BACKGROUND: Few studies have quantified the psychosocial effects of hair loss using standardized instruments in men not seeking treatment for hair loss. OBJECTIVE: Examine self-perception of hair loss and its effects on men from the community. METHODS: Men 18-50 years of age recruited without regard to hair loss, from households near Dayton, Ohio, completed a questionnaire assessing self-perception of hair loss, satisfaction with hair appearance, hair-loss-specific effects and general health status. RESULTS: Men with greater hair loss had more bother, concern about getting older, perceived noticeability to others and greater dissatisfaction with their hair appearance than men with less hair loss. These effects decreased with age for men with hair loss, but regardless of age, perceived noticeability of hair loss increased monotonically with degree of hair loss. CONCLUSIONS: Men with greater hair loss report more negative effects due to their hair loss across all age groups, but the effects were more pronounced in younger men.

Genetic and immunological parameters governing in vivo susceptibility/resistance to retrovirally induced murine malignant histiocytosis.

Malignant histiocytosis sarcoma virus (MHSV) arose as a recombinant of c-Harvey-ras murine sarcoma virus (Ha-MuSV) and Friend mink cell focus-forming virus (F-MCFV). It is a defective acute transforming retrovirus that, along with Friend murine leukemia helper virus (F-MuLV), induces malignant histiocytosis (MH) in susceptible adult mice. We have assessed the in vivo susceptibility to MHSV in inbred homozygous, F1 hybrid, congenic, and recombinant inbred (RI) mice. We have shown that: (1) in vivo resistance to MHSV is multigenic, regulated by MHC and non-MHC genes in a different fashion than with F-MCFV, F-MuLV, or Ha-MuSV; (2) using BXD RI mice, the resistance phenotype is linked with 95.8% probability to two linked loci, Pmv-9 and Iapls3-14, on chromosome 13 (homologous to the area of human chromosome 5 for which a chromosomal break point at position 5q35 is associated with human MH); and (3) CD4+ T cells are critical for MHSV resistance.

Origin and rapid evolution of a novel murine erythroleukemia virus of the spleen focus-forming virus family.

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent Mr of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an Mr of 60,000. During further in vivo passaging, a virus that encodes an Mr-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.

Controlling characteristics of group insureds: risk rating.

Employers seek to manage risk in their group health and life insurance plans through wellness programs and certain hiring practices. In both cases, the Americans with Disabilities Act may ultimately affect the use of these forms of risk rating on the grounds that they are discriminatory.

Approach to a retrovirus vaccine: immunization of mice against Friend virus disease with a replication-defective Friend murine leukemia virus.

In an initial attempt to test the ability of replication-defective retroviruses to immunize against immunologically related pathogenic viruses, we have worked with the erythroleukemogenic Friend retrovirus complex (FV), which consists of a replication-competent helper component, Friend murine leukemia virus (FMuLV), and a related defective pathogenic component, spleen focus-forming virus (SFFV). An 81-base-pair deletion was introduced into the p15E-encoding region of the env gene of an otherwise replication-competent molecular clone of the FMuLV provirus. After transfection of this clone into cells that package the viral RNA in MuLV coats, infectious virus was released into the culture medium. Mouse fibroblasts infected with this virus, here called delta FMuLV, expressed the truncated viral env gene products in their cytoplasm but not on cell surfaces, and culture fluids from these cells did not transmit the infection to fresh mouse fibroblasts. In preliminary experiments, immunization of mice of H-2-congenic BALB/c strains with delta FMuLV conferred levels of immunity to FV disease ranging from weak to relatively strong. Immunized mice developed anti-FV IgM and IgG antibodies and cytotoxic T cells. Mice observed for 15 weeks after the first of two immunizations showed no detectable pathology, but delta FMuLV DNA was detectable in livers of some immunized mice for at least 3-6 weeks. These results suggest that our approach to development of retrovirus vaccines may be a useful one.

Suppression of H-2b-associated resistance to Friend erythroleukemia virus by a class I gene from the H-2d major histocompatibility complex haplotype.

Mice homozygous for the H-2d haplotype at the major histocompatibility complex are markedly more susceptible to erythroleukemia induction by the Friend isolate of murine leukemia retrovirus (FV) than are congenic mice homozygous for the H-2b haplotype. The resistance conferred by the H-2b haplotype is recessive in this cross, since heterozygous F1 mice are as susceptible as parental strain H-2d homozygotes. However, H-2b-associated resistance is not an intrinsically recessive trait, since H-2b/H-2dm1 heterozygotes resemble H-2b homozygotes in their relative resistance to FV; the mutant H-2dm1 haplotype lacks the entire D region of the parental haplotype except for a single class I gene formed by the fusion of its terminal D-region genes to produce a class I gene differing from both parental genes, and thus this finding indicates that one or more D-region genes of the H-2d haplotype can actively suppress H-2b-associated resistance. Unlike H-2dm1, the mutant H-2dm2 haplotype, which retains only the class IDd gene in the D region of the H-2d haplotype, strongly suppresses resistance in H-2b/H-2dm2 heterozygotes, and the presence of Dd as a transgene significantly reduces the resistance of H-2b homozygotes. Since H-2b-associated resistance to FV appears to be due mainly to the capacity of Lb (also called Db), the only class I molecule encoded in the D region of the H-2b haplotype, to present viral epitopes for recognition by FV-specific cytotoxic T lymphocytes, suppression of resistance to FV by the Dd molecule implies that the presence of one class I molecule of the major histocompatibility complex can interfere with either the presentation of viral epitopes by another class I molecule or the generation of T cells that recognize viral epitopes so presented.

Identification of an epitope encoded in the env gene of Friend murine leukemia virus recognized by anti-Friend virus cytotoxic T lymphocytes.

We have previously shown that strong epitopes recognized by anti-Friend virus (FV) cytotoxic T lymphocytes (CTL) in H-2b mice are encoded in both the env and gag/pol regions of the helper friend leukemia virus genome. Two approaches have been used to identify these epitopes. At the nucleic acid level, we have constructed env genes with either of two in-frame deletions: pKR2, an env gene with a 681-bp deletion in the gp70 region and inserted into the pSV2-gpt-1 expression vector; and pKR1, an env gene with an 81-bp deletion in the p15E region and inserted into pSV2-gpt-1. Cell clones were established by transfecting Fisher rat embryo cells with pDb (the H-2Db restriction element), pNEO (for G418 selection) and either pKR1 or pKR2. Db and env gene expression was monitored by immunoprecipitation with polyclonal antibodies or by detection of viral RNA on Northern blots. Expressor cell clones were tested for susceptibility to lysis by polyclonal anti-FV/Db CTL in 51Cr-release assays. Whereas cells expressing pKR1 were lysed to the same extent as cells expressing the intact env gene, cells expressing pKR2 were resistant to lysis, suggesting that all detectable env epitopes are encoded within the 681-bp deletion. Polypeptides representing the two most likely candidate epitopes encoded in this segment were synthesized and tested for their abilities to sensitize FRE cells expressing Db alone for lysis by the CTL. One 17-mer polypeptide, AGTGDRLLNLVQGAYQA [corrected], functioned as a strong CTL epitope in this assay, but the other 18-mer polypeptide was inactive. Studies of the role of this epitope in the immune response to candidate viral vaccines are in progress.

FVB/N: an inbred mouse strain preferable for transgenic analyses.

FVB/N mice offer a system suitable for most transgenic experiments and subsequent genetic analyses. The inbred FVB/N strain is characterized by vigorous reproductive performance and consistently large litters. Moreover, fertilized FVB/N eggs contain large and prominent pronuclei, which facilitate microinjection of DNA. The phenotype of large pronuclei in the zygote is a dominant trait associated with the FVB/N oocyte but not the FVB/N sperm. In experiments to generate transgenic mice, the same DNA constructs were injected into three different types of zygotes: FVB/N, C57BL/6J, and (C57BL/6J x SJL/J)F1. FVB/N zygotes survived well after injection, and transgenic animals were obtained with efficiencies similar to the F1 zygotes and much better than the C57BL/6J zygotes. Genetic markers of the FVB/N strain have been analyzed for 44 loci that cover 15 chromosomes and were compared with those of commonly used inbred strains. In addition to the albino FVB/N strain, pigmented congenic strains of FVB/N are being constructed. These features make the FVB/N strain advantageous to use for research with transgenic mice.

Nonrandom chromosome changes in methylnitrosourea (MNU) induced mouse T-cell lymphomas.

Since nonrandom chromosome changes in neoplastic cells have proven to be good indicators of the site of gene alterations related to transformation, the authors examined the chromosomes of T-cell lymphomas induced in RF/J strain mice with methylnitrosourea (MNU). All treated mice developed thymic lymphomas within 10 weeks of injection. Chromosomes of the thymus cells were examined at intervals before and during lymphoma development, as well as after they were passaged in syngeneic and in nude mice for periods up to 424 days. In preparations made directly from the thymus cells nonrandom numerical and structural alterations were found that involved the X, 3, 15, 4, 8, 12, 14 and 17. (Chromosomes showing alterations are listed in decreasing order of the frequency of their occurrence). In cells passaged in nude mice the chromosomes similarly altered were the 10, X, 3, 12, 6, 1, 4, 19, 15, 18 and 14. In tumor cells passaged in syngeneic mice most of the same chromosomes were involved but the order was 15, 14, X, 1, 5, 6, 3, 11 and 12. The X, 15, 14, 3 and 12 were aberrant in both direct preparations and in those from passaged cells, suggesting that these chromosomes carry genes which, when altered, are particularly important in the multistep process of neoplastic transformation. Most of these chromosomes, or their homologs in other species, have been found to be involved frequently in several different cancers of mice and men, as for example the region on the mouse 15 carrying the Myc and Pvt-1 genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Health care challenges and opportunities in the 1990s.

The 1990s offer both substantial challenges and opportunities for those involved in the delivery of health care. Increasing costs must be managed to ensure that the health of both Americans and America's economy are maintained. Managed care offers the brightest hope for effectively controlling costs while increasing the quality of care.

Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene.

The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) sto stimulate erythroblastosis [Li, J.-P., D'Andrea, A.D., Lodish, H.F. & Baltimore, D. (1990) Nature (London) 343, 762-764]. To directly compare the effects of gp55 with erythropoietin (Epo), we produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemias. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein.

Absence of mouse mammary tumor virus proviral amplification in chemically induced lymphomas of RF/J mice.

RF/J mice are susceptible to the induction of thymic lymphomas by the carcinogens 3-methylcholanthrene and N-methyl-N-nitrosourea. Given the association of mouse mammary tumor virus (MMTV) with certain thymomas, we examined genomic DNA from chemically induced lymphomas of RF/J mice for new MMTV proviruses. Of 13 tissue culture lines derived from 3-methylcholanthrene-induced tumors, 5 had acquired new proviruses. MMTV amplification coincided with the appearance of viral mRNAs and proteins. However, no primary tumors or animal-passaged tumors contained new proviruses. These observations indicate that MMTV does not have a role in the tumor induction process, although it may become activated and amplified in tissue culture lines derived from tumors.

Genetic control of resistance to 3-methylcholanthrene-induced T-cell lymphoma in mice.

Different strains of inbred mice exhibit different levels of susceptibility to T-cell lymphoma induced by the carcinogen 3-methylcholanthrene (MCA). Resistance to MCA-induced lymphoma was dominant over susceptibility in all crosses tested, and in inbred strain mice MCA resistance was found to correlate with resistance to lymphoma induced by a fractionated dose of gamma irradiation. The susceptible RF/J strain was also found to be extraordinarily sensitive to lymphoma induction by low doses of X-irradiation; this low dose sensitivity was also recessive. Experiments on both first and second generation backcross populations supported the hypothesis that a single locus is the main determinant of MCA resistance. Studies examining the possible linkage of low lymphoma incidence to a number of loci failed to establish any clear association, but linkage was seen between the Lyt-2 locus and a significant delay in MCA-induced lymphoma development. We also examined the strain distribution of several activities which might have been expected to have an effect on susceptibility to induced lymphoma. No correlation was seen between resistance and either DNA repair ability, thymic superoxide dismutase levels, or natural killer activity.

FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens.

Immunization of C57BL/6 (B6) mice with FBL, a Friend murine leukemia virus (F-MuLV), induces both tumor-specific cytolytic CD8+ (CTL) and lymphokine-producing CD4+ Th that are effective in adoptive therapy of B6 mice bearing disseminated FBL leukemia. The current study evaluated the F-MuLV antigenic determinants expressed on FBL that are recognized by FBL-reactive CD8+ and CD4+ T cells. To identify the specificity of the FBL-reactive CD8+ CTL, Fisher rat embryo fibroblast (FRE) cells transfected with plasmids encoding F-MuLV gag or envelope (env) gene products plus the class I-restricting element Db were utilized. FBL-reactive CTL recognized FRE target cells transfected with the F-MuLV gag-encoded gene products, but failed to recognize targets expressing F-MuLV env. Attempts to generate env-specific CD8+ CTL by immunization with a recombinant vaccinia virus containing an inserted F-MuLV env gene were unsuccessful, despite the generation of a cytolytic response to vaccinia epitopes, implying that B6 mice fail to generate CD8+ CTL to env determinants. By contrast, CD4+ Th clones recognized FRE target cells transfected with env and not gag genes, and immunization with the recombinant vaccinia virus induced an env-specific CD4+ T cell response. These data show that in a Friend retrovirus-induced tumor model in which tumor rejection can be mediated by either CTL or Th, antigens derived from discrete retroviral proteins are predominantly responsible for activation of each T cell subset.