Immune durability and protection against SARS-CoV-2 re-infection in Syrian hamsters.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a pandemic. As immunity to endemic human coronaviruses (i.e. NL63 or OC43) wanes leading to re-infection, it was unknown if SARS-CoV-2 immunity would also decline permitting repeat infections. Recent case reports confirm previously infected individuals can become re-infected; however, re-infection may be due to heterogeneity in the initial infection or the host immune response, or may be the result of infection with a variant strain that escapes pre-existing immunity. To control these variables, we utilized the Syrian hamster model to evaluate the duration of immunity and susceptibility to re-infection with SARS-CoV-2. Hamsters were given a primary mock or SARS-CoV-2 infection (culture media or 105 TCID50 USA/WA1/2020 isolate, respectively). Mock and SARS-CoV-2 infected hamsters were then given a secondary SARS-CoV-2 infection at 1, 2, 4, or 6 months post-primary infection (n = 14/time point/group). After the primary SARS-CoV-2 infection, hamsters developed anti-spike protein IgG, IgA, and neutralizing antibodies, and these antibodies were maintained for at least 6 months. Upon secondary SARS-CoV-2 challenge, previously SARS-CoV-2 infected animals were protected from weight loss, while all previously mock-infected animals became infected and lost weight. Importantly, despite having high titres of antibodies, one SARS-CoV-2 infected animal re-challenged at 4 months had a breakthrough infection with replicating virus in the upper and lower respiratory tract. These studies demonstrate immunity to SARS-CoV-2 is maintained for 6 months; however, protection may be incomplete and, even in the presence of high antibody titres, previously infected hosts may become re-infected.

Paediatric tracheostomy tubes: recent developments and our current practice.

OBJECTIVE: A variety of paediatric tracheostomy tubes are available. This article reviews the tubes in current use at Great Ormond Street Hospital for Children and Evelina London Children's Hospital. METHODS: This paper outlines our current preferences, and the particular indications for different tracheostomy tubes, speaking valves and other attachments. RESULTS: Our preferred types of tubes have undergone significant design changes. This paper also reports further experience with certain tubes that may be useful in particular circumstances. An updated sizing chart is included for reference purposes. CONCLUSION: The choice of a paediatric tracheostomy tube remains largely determined by individual clinical requirements. Although we still favour a small range of tubes for use in the majority of our patients, there are circumstances in which other varieties are indicated.

Safe balloon sizing for endoscopic dilatation of subglottic stenosis in children.

OBJECTIVES: To describe our experience and provide guidelines for maximum safe balloon sizes according to age in children undergoing balloon dilatation. METHOD: A retrospective review was conducted of children undergoing balloon dilatation for subglottic stenosis in a paediatric tertiary unit between May 2006 and February 2016. RESULTS: A total of 166 patients underwent balloon dilatation. Mean ( ± standard deviation) patient age was 4.5 ± 3.99 years. The median balloon size was 8 mm, the median balloon inflation pressure was 10 atm, and the mean balloon inflation time was 65.1 ± 18.6 seconds. No significant unexpected events occurred. The Pearson correlation co-efficient for the relationship between patient age and balloon size was 0.85 (p = 0.001), suggesting a strongly positive correlation. CONCLUSION: This study demonstrated that balloon dilatation is a safe procedure for airway stenosis. The results suggest using a balloon diameter that is equal to the outer diameter of the age-appropriate endotracheal tube +1 mm for the larynx and subglottis and +2 mm for the trachea.

The incidence and outcome of intracranial haemorrhage in newborns with haemophilia: analysis of the Nationwide Inpatient Sample database.

The incidence of intracranial haemorrhage (ICH) in newborns with haemophilia is unknown. Retrospective studies, estimate the incidence to be around 3%. Because of this uncertainty, we analysed the largest inpatient database in the USA, the Nationwide Inpatient Sample (NIS), to better approximate the incidence of ICH in these patients. ICD-9 coding data were used to reference NIS entries of haemophilia (A, B or C) or von Willebrand's disease (VWD), with intraventricular (IVH), subarachnoid (SAH), subdural (SDH) and/or intraparenchymal (IPH) haemorrhage. Of 9.2 x 10(7) hospitalizations from 1988 to 2001, 11% or 1 x 10(7) were newborns. Of these, 0.00527%, or 580 were diagnosed with haemophilia or VWD. Twenty of 580, or 3.4%, experienced an ICH. The ICH rate in non-haemophilic newborns was 0.11% (P value: <0.0001). The rate of ICH among term haemophilic newborns without sepsis, respiratory distress syndrome (RDS) or congenital heart disease (CHD), delivered without vacuum assist was 1.9%. One death occurred on the day of birth in a term neonate with haemophilia C. The mean length of stay for ICH patients with haemophilia was 28 days (median 28, range: 6-143 days). The mean hospital charges for the group were 102,072 dollars (median 67,551 dollars, range: 9624-467,132 dollars). These data add credence to the estimates of ICH in haemophilic newborns and may guide treatment strategies around the time of their birth. Further, uncomplicated delivery of term, otherwise healthy haemophilic newborns may carry a lesser risk of ICH.

Cooperative role of interferon regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma induces the expression of the indoleamine 2, 3-dioxygenase (INDO) gene in human cells, which plays a role in the inhibitory effect of IFN-gamma on intracellular pathogens and on cell proliferation. Earlier studies established that the IFN-gamma-inducible expression of the INDO gene was dependent on two upstream elements: (i) a 14-base pair sequence homologous to an interferon-stimulated response element (ISRE) sequence found in IFN-alpha-inducible genes and (ii) a 9-base pair palindromic sequence (palindromic element (PE) II) homologous to an interferon-gamma-activated site (GAS) element found in IFN-gamma-inducible genes. A second GAS element (PE I), between ISRE and PE II, was ineffective in supporting a response to IFN-gamma. Studies were carried out to determine the distinction between the two GAS elements and the relative role of the two elements (ISRE and PE II) required for a response to IFN-gamma. The PE I element was able to form a complex with IFN-gamma-activated p91 (STAT1) factor but with lower efficiency than the complex formed with PE II sequence. However, switching the positions of PE I and II sequences in reporter plasmid constructs (containing chloramphenicol acetyltransferase gene) showed that both PE I and PE II were able to support a response to IFN-gamma if located at the position of PE II but not at the position of PE I. Increasing the distance between the ISRE and PE II also affected the level of response, suggesting that the relative position of the two elements is important for optimal stimulus. To explore whether an interaction between the IFN-gamma-regulated factors (IRF-1 and p91) binding to the ISRE and PE II might be important, we tested whether the ISRE sequence could be replaced by another response element, NF-kappaB. The plasmid construct with NF-kappaB element in place of the ISRE was responsive to IFN-gamma, indicating that an interaction between the IRF-1 and p91 factors was not required. The results indicate that the response of INDO gene to IFN-gamma depends on a cooperative role of IFN-gamma-responsive factors binding to the ISRE and GAS elements.

Compatibility and activity of aldesleukin (recombinant interleukin-2) in presence of selected drugs during simulated Y-site administration: evaluation of three methods.

The compatibility and biological activity of aldesleukin (a form of recombinant interleukin-2) in the presence of selected i.v. drugs during simulated Y-site administration was studied. Five milliliters of aldesleukin 33,800 IU/mL in 5% dextrose injection was mixed in glass test tubes with 5 mL of each of 19 i.v. drugs prepared at concentrations used in routine clinical practice. The compatibility of the combinations was assessed by visual examination and spectrophotometry at 0, 0.5, 1, and 2 hours after preparation, and bioassays were conducted to determine the activity of aldesleukin in the combinations. Lorazepam was the only drug visually incompatible with aldesleukin. All the secondary drugs were spectrophotometrically compatible with aldesleukin. However, the bioassays showed that the following drugs reduced the activity of aldesleukin: ganciclovir sodium, lorazepam, pentamidine isethionate, prochlorperazine edisylate, and promethazine hydrochloride. Thus, aldesleukin became less biologically active when combined with four drugs for which visual examination suggested compatibility and when combined with five drugs for which spectrophotometry indicated compatibility. Aldesleukin 33,800 IU/mL in 5% dextrose injection lost significant biological activity in the presence of prochlorperazine edisylate, promethazine hydrochloride, lorazepam, ganciclovir sodium, and pentamidine isethionate during simulated Y-site administration. Visual assessment and spectrophotometry may not be valid methods for assessing possible changes in the biological activity of aldesleukin when combined with other agents.

Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO) gene is implicated in the antimicrobial and antiproliferative effects of IFN-gamma in cell cultures. Earlier studies identified a 96 base pair (bp) regulatory region upstream of the IDO gene that conferred IFN-gamma response to the chloroamphenicol acetyltransferase (CAT) gene linked to herpesvirus thymidine kinase promoter. The IFN-gamma-responsive region was further narrowed to a 67 bp fragment by 3' deletion. This 67 bp fragment contains several sequence elements of potential interest, including a 14 bp sequence homologous to the ISRE sequence found in IFN-alpha-inducible genes and two palindromic sequences (PE I and PE II) homologous to the GAS sequence identified in IFN-gamma-inducible genes. Site-directed mutagenesis studies showed that IFN-gamma-induced expression of IDO-CAT constructs involved cooperation between two elements: the ISRE homolog and the PE II (but not PE I). Either element alone with its flanking sequence was inadequate in conferring an IFN-gamma response to CAT reporter gene. Two IFN-gamma-regulated protein factors interacting with these two elements were identified. The factor binding to the ISRE region was induced with a slower kinetics, required new protein synthesis, and reacted with antibodies to IRF-1. The factor interacting with the PE II region appeared rapidly after treatment with IFN-gamma independently of new protein synthesis, and its binding to DNA probe was blocked by antibodies to p91 factor, reported to bind to GAS element.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.

Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide-lipid interaction. Although the palmitic acid is minimally affected by the peptide, its presence, as suggested by surface balance measurements, results in the establishment of a stable lipid film with DPPC, capable of achieving low surface tension values.

Effect of a bovine lung surfactant protein isolate (SP-B/C) on egg phosphatidylglycerol acyl chain order in a lipid mixture with dipalmitoylphosphatidylcholine and palmitic acid.

Dynamic surface tension measurements of films of a d62 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine:L-alpha-phosphatidyl-DL - glycerol:d31 palmitic acid (d62-DPPC:EggPG:d31-PA) lipid matrix in the presence of a bovine pulmonary surfactant protein isolate (SP-B/C) demonstrate the improved surface activity over that of the lipids alone. Thus, significant interaction of the proteins with the lipid matrix is demonstrated. The effect of SP-B/C on the acyl chain order of the negatively charged EggPG within a d62-DPPC:EggPG:d31-PA lipid matrix in D2O saline was investigated in thermal perturbation Fourier transform IR spectroscopic studies. The EggPG thermotropic phase behavior was determined independently of the other lipid components with perdeuterated lipids and D2O. The data demonstrate the high degree of EggPG acyl chain disorder in the absence of the protein isolate. A broad transition occurs between 30 and 40 degrees C. The addition of the protein isolate did not alter the acyl chain order at 0.281 and 1.46 mg/mL of protein. However, alterations in the lipid carbonyl vibrational mode were observed.

Parenteral formulation development of renin inhibitor Abbott-72517.

Abbott-72517 is an inhibitor of human renin and is being investigated for the treatment of hypertension. It is an orally bioavailable candidate which is being developed for oral as well as intravenous use. The preclinical development of this molecule involved studies to evaluate irritation at the site of injection in an animal model. Several formulation variables such as drug concentration, types of buffer (citrate or acetate), addition of cosolvent (ethanol) to enhance drug solubility, and tonicity modifiers such as glycerin or mannitol were evaluated. Additionally, in vitro formulation--whole blood hemolysis and plasma precipitation studies were conducted. Based on these studies, a liquid formulation containing 1.2 mg/mL Abbott-72517.HCl as base, 0.01M citrate buffer, pH 3.7, in 0.45% sodium chloride containing 2.5% mannitol was recommended for preclinical studies. Various processing and administration parameters were evaluated including filter qualification and compatibility of the drug with typical infusion fluids and administration sets. The liquid formulation was further characterized for physical and chemical stability. It was shown that it has acceptable stability at ambient temperature. Based on the accelerated temperature storage results, T90 at 25 degrees C is > 1 year for the ready-to-use liquid formulation. Additionally, a lyophilized version of the liquid formulation was evaluated.

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

Enhanced gel mobility shift assay for DNA-binding factors.

Gel mobility shift assays are commonly used to study DNA-binding factors involved in the regulation of constitutive, tissue-specific, and inducible genes. We found that addition of 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate (Chaps, a zwitterionic detergent) at relatively high concentration (2.5% or more) to DNA-binding reactions for four different factors (AP-1, SP1, GATA-1, and interferon alpha-regulated factor ISGF3) assayed in cell extracts greatly enhanced the signal for DNA-protein complexes (up to about 20-fold). The amplified signal for DNA-protein complexes so obtained was (at least in part) due to increased binding efficiency, as revealed by greatly reduced amounts of the free probes in the gels. The binding specificity, however, was not compromised. The fact that DNA-protein complex formation with four different factors was stimulated by Chaps suggests that the enhancing effect of Chaps may be more general and not limited to certain types of DNA-binding factors. The results provide the basis for a highly sensitive assay for DNA-binding factors, which may be useful in several types of studies on such factors. Among other detergents tested, Chapso (another zwitterionic detergent), NP-40, and octylglucopyranoside (nonionic detergents) were found also to enhance the complex formation as tested for AP-1 binding, whereas sodium cholate and deoxycholate showed strong inhibition.

Differential regulation of human indoleamine 2,3-dioxygenase gene expression by interferons-gamma and -alpha. Analysis of the regulatory region of the gene and identification of an interferon-gamma-inducible DNA-binding factor.

The induction of indoleamine 2,3-dioxygenase (IDO) activity has been implicated in the antiproliferative action of interferon (IFN)-gamma on tumor cells and the inhibition of intracellular pathogens. Earlier studies have demonstrated that the expression of the IDO gene is induced strongly by IFN-gamma, but very poorly by IFN-alpha despite the presence of a sequence highly homologous to the IFN-alpha-responsive sequence element (interferon-stimulated response element (ISRE)) in its IFN-gamma-responsive control region. In addition, a sequence with a partial homology to the IFN-gamma-responsive sequence (GAS) identified by Lew et al. (Lew, D.J., Decker, T., Strehlow, I., and Darnell, J.E., Jr. (1991) Mol. Cell. Biol. 11, 182-191) in a human gene for a guanylate-binding protein and to the X box sequence found in all major histocompatibility complex class II genes was found. Deletion experiments have indicated that the ISRE homolog (but not the GAS-related or the X box-related sequence) was essential for an inducibility by IFN-gamma. To investigate the lack of inducibility by IFN-alpha despite the presence of an ISRE homolog, the binding of this ISRE homolog to the IFN-alpha-stimulated gene factor 3 (ISGF3) was examined. Gel mobility shift experiments and competition experiments indicated that this ISRE homolog did not form a stable complex with ISGF3. This may account for a poor inducibility by IFN-alpha. This inability to bind ISGF3 appears to be (at least in part) due to minor differences between the nucleotide sequence of the ISRE homolog present in the IDO gene promoter and the ISRE consensus sequence found in IFN-alpha-inducible genes. An IFN-gamma-inducible DNA-binding factor was identified with characteristics different from ISGF3: (i) the IFN-gamma-inducible factor was detected in the nuclear extracts, but not in the cytoplasmic extracts; and (ii) the appearance of this DNA-binding factor required new protein synthesis, which could explain the dependence on new protein synthesis for the induction of IDO by IFN-gamma.

Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-->p11 by fluorescent in situ hybridization.

Indolamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase are two enzymes that degrade tryptophan to N-formylkynurenine. The gene (TDO2) for tryptophane 2,3-dioxygenase has been localized to 4q31-->32. We now report localization of INDO (the gene encoding indolamine 2,3-dioxygenase) by fluorescent in situ hybridization to 8p12-->p11.

Analysis of interferon-gamma resistant mutants that are possibly defective in their signaling mechanism.

Our previous observations indicated that mutants partially resistant to IFN-gamma cytotoxicity were defective in the induction of indoleamine 2,3-dioxygenase, (IDO). Two mutants highly resistant to IFN-gamma were isolated following a second round of mutagenesis. The resistance to IFN-gamma was inversely correlated with the inducibility of IDO in these mutants. Moreover, several other IFN-gamma responsive genes, including those encoding 2-5A synthetase, GTP cyclohydrolase and HLA-DR alpha, were also differentially altered in their expression upon INF-gamma treatment. IFN-gamma receptor gene expression was not changed nor was the binding of the receptor to IFN-gamma. Southern blot analysis failed to reveal any significant abnormality in the IDO gene structure in these mutants. We therefore suggest that these mutants are defective in the IFN-gamma signaling pathway and will be useful in further analysis of the biochemical mechanism of IFN-gamma activated gene expression in target cells.

Regulation of indoleamine 2,3-dioxygenase gene expression in human fibroblasts by interferon-gamma. Upstream control region discriminates between interferon-gamma and interferon-alpha.

The interferon (IFN)-gamma-mediated induction of indoleamine 2,3-dioxygenase (IDO) enzyme, which converts tryptophan into N-formylkynurenine, has been implicated in the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, and in the antiproliferative effect of IFN-gamma on tumor cells. The IDO activity is induced strongly in many cell types by IFN-gamma but rather poorly by IFN-alpha or -beta. A genomic DNA clone containing part of the transcribed region of the IDO gene and approximately 13 kilobases (kb) of the 5'-upstream DNA sequence was isolated and analyzed. An approximately 1.4-kb fragment of this clone, containing 329 nucleotides of the transcribed sequence and approximately 1.1 kb of the 5'-upstream sequence, when ligated to chloramphenicol acetyltransferase (CAT) structural gene made its expression inducible by IFN-gamma, but this construct responded poorly, if at all, to IFN-alpha 2. Deletion constructs derived from this plasmid narrowed down the IFN-gamma-responsive region to a 151-nucleotide segment (-495/-344) which also contained a 14-nucleotide sequence (GGTTTCAGTTTTCC) highly homologous to the IFN(alpha)-stimulated response element (ISRE) that has been found so far in all cellular genes inducible with IFN-alpha or -beta. Expression of CAT activity was stimulated by IFN-gamma more effectively than by IFN-alpha 2 when a 155-nucleotide fragment (-495/-340) containing the 151-nucleotide segment required for IFN-gamma response was inserted before herpes simplex virus thymidine kinase promoter linked to CAT structural gene. The results indicate that despite the presence of an ISRE, the control region of the IDO gene can distinguish between IFN-gamma and IFN-alpha. This may account for the differential activation of IDO gene expression by IFN-gamma as against IFN-alpha or -beta in intact cells, and suggests that the response of ISRE to IFN-alpha or -beta may be governed by other features in the upstream control region of this gene.

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA.

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.

Regulation of cellular gene expression by interferon-gamma: involvement of multiple pathways.

Interferons (IFNs) classified as type I (IFN-alpha and -beta) and type II (IFN-gamma) interact with different receptors and regulate the expression of a number of genes in common, whereas the expression of certain other genes is regulated differentially by the type I or type II IFNs. Regulation of cellular gene expression by IFN-gamma was studied with the help of two cDNA clones (called C5-4 and C13) isolated in our laboratory and cDNA clones for human leukocyte antigen (HLA) class I (HLA-B) and class II (HLA-DR alpha, -DR beta) genes. The results indicate that IFN-gamma induced the expression of the cognate genes, but in different manners. IFN-gamma induced the transcription of all four genes as determined by nuclear run-on transcription analyses, but the induction of C5-4 genes transcription was inhibited by cycloheximide and anisomycin, indicating that some newly synthesized protein, presumably induced by IFN-gamma, was required for the transcriptional activation of the C5-4 gene. On the contrary, IFN-gamma-induced transcription of HLA class I, class II and C13 genes was unaffected by cycloheximide or anisomycin. However, these inhibitors completely blocked the accumulation of the HLA class II gene transcripts, but not HLA class I or C13 gene transcripts. Results suggest that some newly synthesized protein factor was required for IFN-gamma-induced accumulation of HLA class II gene transcripts and played a role at a step subsequent to the transcriptional activation by IFN-gamma. Evidence was obtained which suggests that the putative protein factor(s) required was induced by IFN-gamma. These studies indicate that IFN-gamma regulates the expression of cellular genes through multiple pathways.