The long-term outcome of interstitial lung disease with anti-aminoacyl-tRNA synthetase antibodies.

RATIONALE: Anti-aminoacyl transfer RNA synthetase antibodies (anti-ARS) are a group of myositis-specific autoantibodies that are detected in the sera of patients with polymyositis and dermatomyositis (PM/DM) and also in those of patients with idiopathic interstitial pneumonias without any connective tissue disease (CTD), including PM/DM. Although we reported the clinical characteristics of interstitial lung disease with anti-ARS antibodies (ARS-ILD) with and without PM/DM, the long-term prognosis of ARS-ILD remains undetermined. As our previous studies revealed that ARS-ILD without PM/DM was similar to CTD-associated ILD, and that ARS-ILD with PM/DM was radiologically suggestive of a nonspecific interstitial pneumonia (NSIP) pathological pattern, we hypothesized that the prognosis of ARS-ILD might be distinct from that of idiopathic pulmonary fibrosis (IPF) without anti-ARS. OBJECTIVES: To elucidate the long-term outcome of ARS-ILD with and without PM/DM and compare it to that of IPF. METHODS: A two-center retrospective study was conducted. The study population comprised 36 patients with ARS-ILD (8 with PM, 12 with DM, and 16 without myositis throughout the course), 100 patients with IPF without anti-ARS, and 7 patients with NSIP without anti-ARS. The presence of anti-ARS was determined by RNA immunoprecipitation using the sera obtained at the time of diagnosis before specific treatment. MEASUREMENTS AND MAIN RESULTS: During the observational period (median 49 months; range, 1-114 months), 7 patients with ARS-ILD (19%; 3 with PM, 1 with DM, and 3 without PM/DM) and 51 patients with IPF (51%) died. Patients with ARS-ILD had better overall survival than those with IPF (log-rank test, P < 0.001) and similar survival compared to those with NSIP (log-rank test, P = 0.59). The prognosis for patients with ARS-ILD was similar between those with and without myositis (log-rank test, P = 0.91). At the median follow-up time of 76.5 months, 14 of the 36 patients with ARS-ILD had deteriorated. Both a decline in forced vital capacity or an initiation of long-term oxygen therapy during the course (odds ratio [OR], 5.34) and acute exacerbation (OR, 28.4) significantly increased the mortality risk. CONCLUSIONS: The long-term outcome of ARS-ILD was significantly better than that of IPF regardless of the presence or absence of myositis.

Nonclinical Safety Testing of RNA Vaccines.

In this chapter, we first consider the overall goal of nonclinical safety testing during drug development and have a brief overview of its regulatory background. We then discuss some basic requirements of safety/toxicity testing before concentrating on the safety testing of RNA vaccines and developing a sample RNA vaccine safety testing program.

An unexpected role for RNA-sensing toll-like receptors in a murine model of DNA accrual.

OBJECTIVES: The goal of this study was to determine whether endosomal Toll-like receptors (TLRs) contribute to the clinical manifestation of systemic autoimmunity exhibited by mice that lack the lysosomal nuclease DNaseII. METHODS: DNaseII/IFNaR double deficient mice were intercrossed with Unc93b13d/3d mice to generate DNaseII-/-mice with non-functional endosomal TLRs. The resulting triple deficient mice were evaluated for arthritis, autoantibody production, splenomegaly, and extramedullary haematopoiesis. B cells from both strains were evaluated for their capacity to respond to endogenous DNA by using small oligonucleotide based TLR9D ligands and a novel class of bifunctional anti-DNA antibodies. RESULTS: Mice that fail to express DNaseII, IFNaR, and Unc93b1 still develop arthritis but do not make autoantibodies, develop splenomegaly, or exhibit extramedullary haematopoiesis. DNaseII-/- IFNaR-/- B cells can respond to synthetic ODNs, but not to endogenous dsDNA. CONCLUSIONS: RNA-reactive TLRs, presumably TLR7, are required for autoantibody production, splenomegaly, and extramedullary haematopoiesis in the DNaseII-/- model of systemic autoimmunity.

Targeted deletion of the gene encoding the La autoantigen (Sjögren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss.

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1(Cre) La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.

The impact of preoperative immunonutrition and other nutrition models on tumor infiltrative lymphocytes in colorectal cancer patients.

AIM: The importance of the alteration of tumor infiltrative lymphocytes (CD4(+), CD8(+), CD16(+), and CD56(+)) in colorectal cancer prognosis is well known. In this study, we analyzed the effect of preoperative immunonutrition and different nutritional models on the clinical condition of colorectal cancer patients. METHODS: Twenty-eight colorectal cancer patients were grouped into 4 groups according to their nutrition regimens randomly and were given immunonutrition (IMN), standard enteral (SE), total parental nutrition (TPN), and normal nutrition (NN) regimens, all of which contained the same calorie-nitrogen content within a 7-day preoperative period. All patients had an endoscopic biopsy before and after the regimen, and the lymphocyte population infiltrating mucosal parts of the resected tumor tissue were evaluated. Immunohistochemical analysis of the tissue specimens was performed by staining with antihuman CD4(+), CD8(+), CD16(+), and CD56(+) antibodies. RESULTS: After nutrition, there were significant increases in each of the 4 groups of CD4(+) and CD8(+) cells within the tumor. Comparing the rates of augmentation, the increased rates of the CD8(+) cells infiltrating the tumor after nutrition in the patients who were fed with IMN were significantly more than the ones in other groups (P = .01). CD16(+) cell infiltration was significantly higher in all groups except the SE and IMN groups. The SE group had increased CD56(+) cell infiltration compared with the other groups. CONCLUSIONS: In the colorectal cancer patients who had nutrition in the 7-day preoperative period, except for the SE nutrition group, there were significant increases of infiltration of CD56(+) cells at the mucosal part of the tumor tissue within the CD4(+) and CD8(+) cell population. When postnutrition values were compared, there was a marked increase of CD8(+) cells in the IMN group.

Anti-DNA:RNA antibodies and silicon photonic microring resonators: increased sensitivity for multiplexed microRNA detection.

In this paper, we present a method for the sensitive detection of microRNAs (miRNAs) utilizing an antibody that specifically recognizes DNA:RNA heteroduplexes and a silicon photonic microring resonator array transduction platform. Microring resonator arrays are covalently functionalized with DNA capture probes that are complementary to solution phase miRNA targets. Following hybridization on the sensor, the anti-DNA:RNA antibody is introduced and binds selectively to the heteroduplexes, giving a larger signal than the original miRNA hybridization due to the increased mass of the antibody, as compared to the 22-mer oligoribonucleotide. Furthermore, the secondary recognition step is performed in neat buffer solution and at relatively higher antibody concentrations, facilitating the detection of miRNAs of interest. The intrinsic sensitivity of the microring resonator platform coupled with the amplification provided by the anti-DNA:RNA antibodies allows for the detection of microRNAs at concentrations as low as 10 pM (350 amol). The simplicity and sequence generality of this amplification method position it as a promising tool for high-throughput, multiplexed miRNA analysis as well as a range of other RNA based detection applications.

Vitamin a supplements alleviate inflammatory responses in reproductive tracts of male mice infected with pseudorabies virus.

Vitamin A is largely thought to have immune potential for mammal health; however, no conclusive mechanisms exist regarding its role in the pathogen-initiated innate immune response, or in the linkage between the innate and adaptive immune system during sperm formation in the male reproductive tract. Therefore, this study was conducted to evaluate the nutritional protective effect of vitamin A supplementation on reproductive performance and immune function of the male mouse challenged with pseudorabies virus (PRV). Sperm quality, testis toll-like receptors (TLRs) mRNA expression levels, and serum concentration of cytokines and immunoglobulins at 7 or 14 days post-injection were compared between control mice and PRV-challenged mice fed the same diet supplemented with vitamin A at 0, 4000, 10,000, 25,000 and 50,000 IU/kg, respectively. PRV- and phosphate buffered saline (PBS)-injection were performed when the mice in the unsupplemented group were marginally deficient in vitamin A. Sperm quality (sperm density and deformity ratio) of PRV-injected mice was significantly harmed by PRV, but this effect was attenuated by increased vitamin A consumption. Vitamin A supplements also attenuated PRV-challenge-induced increase in testis TLR3, TLR7, and TLR9 mRNA expression and serum pro-inflammatory cytokine (gamma interferon, IFN-gamma; and interleukin 1-beta,IL-1beta) concentration, and decrease in serum anti-inflammatory cytokine (IL-10) concentration. Higher than normal vitamin A consumption was recommended to counteract the deleterious effects of viral invasion, possibly through the downregulated expression of TLRs, and thus to improve immunity and reproductivity of male mice challenged with an invading pathogen.

Innate immune recognition of nucleic acids.

The innate immune system employs a number of pattern recognition receptor families in response to DNAs and RNAs, either from invading microbes or within the hosts. These include the Toll-like receptors (TLRs), the retinoic acid inducible gene I (RIG-I) like receptors (RLRs), and the nucleotide-binding domain leucine-rich repeat/NOD-like receptor (NLRs), among other potential sensors in the cytoplasm. These receptors are composed of modular domain architecture, with ligand binding/sensing domains and signaling domains regulated either through dimerization/oligomerization, or conformational changes directed by enzymatic activities. Signaling pathways from different families of receptors converge on their respective common adapter proteins and lead to activation of transcription factors or caspases. Many of these receptors induce orchestrated responses to similar ligands from different cell types, resulting in redundant and complementary immunity to infections. This highly efficient defense system is a double-edged sword: inappropriate reaction to host ligands leads to compromised innate tolerance and autoimmune diseases. Structural studies of innate immune receptors and their signaling pathways are essential in our understanding of pattern recognition mechanisms and design of more efficient vaccine adjuvants.

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles, a novel class of immunomodulators.

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.

Immunoenhancement via enteral nutrition.

Arginine, glutamine, the long chain polyunsaturated omega-3 and omega-6 fatty acids, and, to a lesser extent, ribonucleic acid and the vitamins E, C, and A have pharmacologic effects when given in amounts in excess of what is needed to prevent nutritional deficiency. These effects are exerted primarily via the immune system, and immunoenhancing diets that embody the recently developed principles of nutritional pharmacology have been shown to reduce infectious complications by approximately 75% in surgical patients and hospital stay by more than 20% in surgical patients and patients in the intensive care unit in three independent, prospective, randomized studies, two of which were double-blinded. These findings suggest that specialized diets can be designed that will be of benefit to patients with cancer, atherosclerosis, intestinal diseases, autoimmune diseases, infections, and trauma. However, the interaction of these nutrients in pharmacologic amounts with standard pharmacologic drugs is largely unknown, as are the effects of long-term administration of specialized diets to treat these conditions.

Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue.

The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissue-specific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of expression of multiple genes encoding calmodulin during spermatogenesis.

It has recently been determined that the intracellular calcium receptor, calmodulin, is encoded by a multigene family. At least three calmodulin genes that encode the identical protein are expressed in adult testis from a variety of mammalian species. The complementary techniques of RNA blot hybridization and in situ hybridization were used to assess the relative levels of calmodulin RNAs during rat testis cell differentiation. RNA isolated from highly purified populations of somatic or germinal cells and sections of fixed testis were analyzed for hybridization to specific probes corresponding to the three separate rat calmodulin genes. The level of each calmodulin RNA can be described by a unique developmental pattern during spermatogenesis. The steady state levels of transcripts corresponding to calmodulin gene I (CaM I) and III increase in early meiotic cells (leptotene-zygotene spermatocytes) and remain constant throughout meiosis. The level of the CaM II RNA increases between early and mid-pachytene spermatocytes, but is only transiently elevated. The level of this RNA decreases in round spermatids and is almost nondetectable in cytoplasts shed from elongating spermatids. The levels of CaM I RNAs are maintained in early round spermatids, but only RNA derived from the CaM III gene is still evident in late spermatids. The data indicate that an individual germ cell contains RNAs derived from multiple calmodulin genes and suggests that the divergent calmodulin genes may respond to different cellular regulatory signals.

Immune modulators as antiviral agents.

Immune modulators have exhibited some limited efficacy in the prevention or treatment of viral infection. Such agents have included transfer factor, thymosin, thymic humoral factors, levamisole, isoprinosine, immune RNA, young lymphocytes, vitamin C, and BCG. This article focuses on data that have examined clinical application of immune adjuvant therapy.

Acrylamide gel analysis of sucrose gradient-derived RNA B fractions transferring delayed sensitivity in vitro.

Sucrose-derived RNA fractions transferring specific delayed sensitivity in vitro were extracted from mono-(p-azobenzenearsonate)-N-chloracetyl-L-tyrosine (ARSNAT)- or keyhole limpet hemocyanin (KLH)-sensitized guinea pigs. Fractions having biological activity were assessed by acrylamide gel analysis to enumerate the number of RNA species in active fractions, and to compare and examine the banding patterns of each RNA fraction. Each isolated B fraction of RNA exhibited multiple bands of RNA with molecular weights ranging from 4.0 X 10(5) to 8.5 X 10(5). Depending on the source and antigen sensitivity of the RNA donor, several differences were observed among the analyzed fractions. These were bands of varying intensity, presence of additional RNA bands, absence of bands in certain positions, and RNA bands migrating in different positions on the gels. Acrylamide gel analysis separation, and resolution of B fractions with specific immunobiological activity now offers an approach for further isolation and resolution of the active species.

[High-polymeric xenogenic RNA as an antitumor immunity stimulator]. / Vysokopolimernaia ksenogennaia RNK kak stimuliator protivoopukholevogo immuniteta.

To immunize CC57BR mice a suspension of live cells of Krebs-2 ascites tumour was administered intradermally into the tail partially amputated afterwards. The growth of the tumour transplanted intraperitoneally was inhibited by 23% only after twofold immunization. Single immunization with tumour cell incubated with the cattle liver RNA preparation in conjunction with intraperitoneal administration of RNA following tumour transplantation inhibited its growth by 43--53%, while twofold administration by 84--88%. The high polymeric fraction of the preparation enhanced the immunization effect to the same measures the initial overall preparation. The treatment of the preparation with RNAase and partial depolymerization of RNA in the course of isolation resulted in the activity loss. It is concluded that the capacity of the RNA preparation for stimulating antitumour immunity is due to high polymeric fraction of RNA.

Ribavirin treatment in murine autoimmune disease. I. Therapeutic efficacy and effect on the immune response.

NZB/W F1 female mice were treated from 20 weeks of age with ribavirin (a broad spectrum antiviral drug), cyclophosphamide, or saline. Treatment with ribavirin (250 mg/kg twice weekly) prolonged survival from 9.8 to 18.5 months, reduced anti-DNA antibodies, and prevented proteinuria. Ability of ribavirin to prolong survival was dose related when given on a twice weekly schedule. However, daily ribavirin (25 mg/kg/day) was as effective as higher intermittent doses. Optimal ribavirin therapy was equal to cyclophosphamide treatment with regard to prolongation of survival. Ribavirin treatment did not significantly alter the body weight, hematocrit, WBC count, serum immunoglobulins, or Coombs reactivity. No alterations in either cellular or humoral immune responses were noted in NZB/W F1 or BALB/c mice treated for prolonged periods with ribavirin. The impressive therapeutic response to a broad spectrum antiviral agent seen in mice already manifesting immune complex nephritis provides a new therapeutic approach to the treatment of autoimmunity.

[Effect of adjuvants on the cellular function of the monocytic phagocytosing system]. / Vliianie ad"iuvantov na funktsiiu kletok monotsitarnoi fagotsitiruiushchei sistemy

The authors studied the effect of adjuvants differing by origin and physico-chemical nature (complete Freund's adjuvant, S. typhi endoxin, cadmium sulfate, iron trichloride) on the ingestion and digestion of erythrocytes of the sheep by the cells of monocytic phagocytizing system, on the persistence of the antigen in these cells, its complexation with the RNA-macrophages and the function of their lysosomal apparatus. The adjuvants change the phagocytizing capacity of the macrophages only in their administration in vivo. Administration to the animals of Freund adjuvant and of the S. typhic endotoxin somewhat increased the ingestion of the antigens, whereas the administration of FeCl3 and CdSO4 failed to change it or even somewhat decreased it. The capacity of ingestion of the antigen in vitro in macrophages obtained from the animals treated with the adjuvants was changed in comparison with the normal. All the adjuvants tested produced a marked action on the lysosomal apparatus of the cells of the monocytic phagocytizing system: they changed the activity of catepsin, promoted the accumulation and the retention in the lysosomes of the highly immunogenic fractions of the antigen, and increased the permeability (except the CdSO4 of the lysosomal membranes in the cells of the antigen binding with the RNA of the cells of the peritoneal exudate or the splenic cells.

Safety assessment of poly I:C in NZB/NZW mice (38565).

The present report confirms the findings by Steinberg et al. (9) that repeated intraperitoneal injections of poly I:C (3 mug/g, three times per wk, 40-52 doses) enhanced the incidence and severity of glomerular lesions that occur spontaneously in NZG/NZW mice and also increased the development of circulating antibody against nucleic acids. This effect was minimal when only six intraperitoneal doses were given in 1 mug/g amount at weekly intervals. Intranasal administration of poly I:C (0.2 mug/g, three times per wk, 40 doses) or six doses of the drug (1 mug/g weekly) caused no apparent potentiation of glomerular response. ICR/Ha mice, which do not suffer from the spontaneously occurring disease, were uneffected by poly I:C treatment except for occasional development of antibody against poly I:C or DNA.