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In a recent expert meeting, Swedish recommendations for the treatment of hepatitis C virus (HCV) infection were updated. An interferon-free combination of direct-acting antiviral agents is considered and indicated for all patients with chronic HCV infection, but the ability to treat all is limited primarily by high cost of medication. The group of patients prioritized for therapeutic intervention has been extended to also include fertile women desiring to become pregnant. A more thorough discussion of treatment for people who inject drugs (PWIDs) in order to diminish transmission is included, and the clinical significance of baseline NS5A resistance associated variants (RAVs), also known as resistance associated substitutions (RASs), for the treatment of HCV genotype 1a or 3 infection is discussed.

The modern medical treatment of HIV with antiretroviral therapy (ART) has drastically reduced the morbidity and mortality in patients infected with this virus. ART has also been shown to reduce the transmission risk from individual patients as well as the spread of the infection at the population level. This position statement from the Public Health Agency of Sweden and the Swedish Reference Group for Antiviral Therapy is based on a workshop organized in the fall of 2012. It summarizes the latest research and knowledge on the risk of HIV transmission from patients on ART, with a focus on the risk of sexual transmission. The risk of transmission via shared injection equipment among intravenous drug users is also examined, as is the risk of mother-to-child transmission. Based on current knowledge, the risk of transmission through vaginal or anal intercourse involving the use of a condom has been judged to be minimal, provided that the person infected with HIV fulfils the criteria for effective ART. This probably also applies to unprotected intercourse, provided that no other sexually transmitted infections are present, although it is not currently possible to fully support this conclusion with direct scientific evidence. ART is judged to markedly reduce the risk of blood-borne transmission between people who share injection equipment. Finally, the risk of transmission from mother to child is very low, provided that ART is started well in advance of delivery.

Objective: To investigate the feasibility of ritonavir-boosted atazanavir monotherapy in HIV-1-infected patients with stable antiretroviral therapy (ART). Design: Single-armed single-center pilot trial. Methods: Adult HIV-1-infected patients, without protease inhibitor (PI) experience, were eligible if they had maintained a viral load

In a recent expert meeting, Swedish recommendations for the treatment of HCV infection were updated. An interferon-free combination of direct-acting antiviral agents was recommended as the first line standard-of-care treatment for chronic HCV infection. Interferon-based therapy should be considered as a second line option after an individual benefit-risk assessment. Treatment is strongly recommended for HCV infected patients with bridging fibrosis or cirrhosis (Metavir stages F3–4), before and after liver transplantation, and in the presence of extra-hepatic manifestations. Additionally, patients with moderate liver fibrosis (stage F2) as well as women in need of in vitro fertilisation should be prioritised for therapeutic intervention. Treatment indications for people who inject drugs, children, chronic kidney disease and HIV co-infection are also discussed.

E2F1 is a crucial downstream effector of the retinoblastoma protein (pRB) pathway. To address the consequences of short-term increase in E2F1 activity in adult tissues, we generated transgenic mice expressing the human E2F1 protein fused to the oestrogen receptor (ER) ligand-binding domain. The expression of the ER-E2F1 fusion protein, which is inactive in the absence of 4-hydroxy tamoxifen (OHT), was targeted to the testes. We show that short-term activation of E2F1 results in activation of E2F target genes and apoptosis of germ cells. Consistent with our previously published results, the apoptotic response was independent of p53. Persistent E2F1 activation for 3 weeks led to massive apoptosis and severe testicular atrophy with seminiferous tubules containing only Sertoli cells and clusters of undifferentiated spermatogonia. The latter showed high expression of ER-E2F1 and excessive mitotic activity, including atypical mitoses. In addition, gonocyte-like dysplastic germ cells, resembling carcinoma in situ (CIS) cells in humans, appeared. Our results show that a relatively short period of deregulated E2F1 activity in testicles can induce premalignant changes. Moreover, we demonstrate the feasibility of tissue-specific expression of conditional ER-E2F1 in transgenic mice.

Clostridium difficile-associated diarrhea (CDAD) is regarded as an emerging nosocomial infection. All patients positive for C. difficile in Sweden were recorded during 1995, including primary care patients. Those positive for toxin in feces were defined as CDAD cases. A total of 5,133 CDAD cases were recorded (58 per 100,000 inhabitants per year), as compared with 86 cases diagnosed in 1978 and 553 in 1983. CDAD was almost twice as prevalent as all (combined) diagnosed domestic cases of reportable bacterial and protozoal diarrhea. The age-specific incidence was little affected by gender but increased > 10-fold over the age range of 60-98 years. The differences in overall CDAD incidence were sixfold between counties and threefold between major hospitals. Among hospitalized patients the incidences were highest in geriatric/rehabilitation wards, followed by infectious diseases and internal medicine wards; 28% of all cases involved no recent hospitalization and were defined as community-acquired CDAD.

In a recent expert meeting, Swedish recommendations for the treatment of HCV infection were upgraded. The panel recommends vaccination against both hepatitis A and B in patients with HCV. Therapy for symptomatic acute HCV infection should be initiated if spontaneous resolution has not occurred within 12 weeks, whereas asymptomatic acute HCV should be treated upon detection. Patients with genotype 2/3 infection should generally be treated for 24 weeks. In patients with a very rapid viral response (vRVR), i.e. HCV RNA below 1000 IU/ml on d 7, treatment can be shortened to 12-16 weeks, provided that no dose reduction has been made. For genotype 1 patients with rapid viral response (RVR), 24 weeks treatment is recommended. For patients with a complete early viral response (cEVR), 48 weeks treatment is recommended, whereas 72 weeks treatment should be considered for patients with partial early viral response (pEVR). For patients with difficult-to-treat disease and with pronounced anaemia, erythropoietin can be used to maintain the ribavirin dose. In HCV-HIV coinfected patients, combination therapy for HCV should, if possible, be initiated before anti-retroviral therapy (ART) is indicated. For liver transplant patients pre-emptive therapy is not recommended; hence, treatment should be deferred until histological recurrence.

We studied the HIV-1 load and nelfinavir (NFV) concentrations in cerebrospinal fluid (CSF) after long-term successful NFV-based therapy, using ultrasensitive methods of detection. 19 patients without virological failure in plasma, who had been treated with 2 nucleoside analogue reverse transcripaste inhibitors (NRTI) and NFV for a minimum of 18 months were included. HIV-RNA was determined in plasma and CSF using an ultrasensitive method (2 copies/ml). Total and free concentrations of NFV were analysed using high liquid chromatography with UV-light detection. 12 out of 19 (63%) patients had2 copies HIV-RNA/ml in CSF. Seven subjects ranged between 3 and 39 copies/ml, 2 of whom had a slightly higher viral load in CSF than in plasma. NFV was detected in CSF in 16 out of 18 patients analysed and was quantifiable in 8 patients, at concentrations ranging from 6 to 29 nM. There was no correlation between NFV concentration and HIV-RNA levels. Long-term therapy with NFV + 2 NRTI showed no increased rate of virological treatment failure within the central nervous system (CNS) in compliant patients, despite earlier reports of lack of NFV penetration to CNS. Using a highly sensitive method, NFV was detected and quantified in the CSF, although at low values, which could have contributed to the high anti-HIV-1 efficacy of the therapy seen in our subjects.

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)2 vitamin D3 significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 μM), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.

The energy source shift-down described in the preceding paper (Molin et al., J. Bacteriol. 131: 7–17, 1977) was used to study the effects of shift-down on protein synthesis. The overall rate of protein synthesis was reduced immediately, and to the same extent, in stringent and relaxed strains. The primary effect of the shift was a slowing down of the polypeptide chain growth rate, a finding not previously reported. In stringent strains the normal, preshift rate was reestablished within 2 to 3 min, whereas in relaxed strains the chain growth rate remained low for about 20 min before slowly returning to the normal value, which was reestablished some 50 to 60 min after the shift. Throughout this transition, the stability of messenger ribonucleic acid (mRNA) remained unchanged in both strains. We interpret these findings as evidence of the more rapid reduction of the mRNA pool in the stringent strain after shift-down: we believe that very soon after the shift, the stringent strain reduces its pool of mRNA and with it the number of ribosomes engaged in protein synthesis. In this manner the number of active ribosomes is adjusted to the availability of energy and carbon. The relaxed strain cannot rapidly reduce its mRNA pool, which thus remains large enough to engage a near-preshift number of ribosomes during a prolonged period; as a consequence its ribosomes must work at a reduced rate. The possibility that ppGpp is involved in the control of mRNA production is discussed. After shift-down, the initial part of β-galactosidase (the auto-α fragment) was produced at a higher rate than complete β-galactosidase in the relaxed strain, as expected when translation is impeded.

Ornithine transcarbamylase formation in Escherichia coli strain B is known to be increased by excess arginine. Partial de-repression, however, occurs by growth in an enriched but arginine-free medium or by arginine-limited growth in a chemostat. Furthermore, at growth temperatures higher than 39 °C excess arginine no longer increases but actually represses enzyme formation in strain B, while temperature does not affect control in a repressible strain like K or in a constitutive mutant from B. These physiological effects are intrinsic to the argR alleles of B and K and not to other strain characteristics and suggest that arginine formation is controlled by repression in both strains B and K, but that a secondary effect of arginine produces apparent induction in strain B. It is postulated that the repressor product of argR B gives rise, depending on the arginine concentration, to forms with different affinities for the operator site.

In order to find effective methods to isolate microorganisms that excrete large amounts of amino acids, a number of mutants of Escherichia coli ATCC 4157 were prepared and characterized. The majority were isolated for resistance to amino acid analogs. The penicillin method was also employed to select potential producer strains devoid of a biosynthetic enzyme in one case and a catabolic enzyme (tryptophanase) in another. Both of these enzymes were suspected of reducing the yield of an excerted amino acid. The identification of amino acid excreting colonies was aided by the use of pigmented auxotrophic test bacteria. Amino acid analog resistance was frequently found to increase amino acid production. In particular, excretion of valine and glutamic acid could be achieved with comparative ease. Conditions of culture were of extreme importance for the amount and kind of amino acid produced. Under certain conditions a norvaline-resistant strain and even better a leucine-requiring derivative of it excreted more than 2 mg./ml. of valine into the culture medium. The acetolactate-forming enzyme of this strain was not repressible by leucine in contrast to the same enzyme in the wild type. Reversal of the growth inhibitory effects of amino acid analogs by amino acids was studied on agar plates. This method gave too complex results to be a simple guide in the selection of analogs suitable for the isolation of a particular type of production mutant.

The present experiments utilize a mutant of E. coli B lacking deoxyribonucleoside-catabolizing enzymes. This strain, OK441, has mutations affecting the following enzymes : thymidine phosphorylase, purine nucleoside phosphorylase, cytidine deaminase, and adenosine deaminase. The results with OK441 show that E. coZi, in contrast to lactobacilli and mammalian cells, does not incorporate any deoxyribonucleoside other than thymidine into its DNA. E. coli may be unable to phosphorylate deoxycytidine, deoxyadenosine, and deoxyguanosine. In agreement with this suggestion, only thymidine kinase but no other deoxyribonucleotide kinase could be detected in extracts of OK441. pounds.

The dnaF mutant of Escherichia coli has been shown to contain very low ribonucleoside diphosphate reductase activity. The B1 sub unit of the reductase is heat-sensitive in the mutant.

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.

We have constructed thermoinducible plasmids carrying the gene (dut) for the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli. A 9.4-kb BamHI restriction enzyme fragment carrying the dut gene was inserted into the runaway-replication plasmid pKN402A (Uhlin et al., Gene 6 (1979) 91-106). Strains carrying such plasmids increased their dUTPase activity considerably. In minimal medium a 200-fold increase was demonstrated. A smaller (1.5-kb) SacI-BamHI fragment from the dut region was also cloned into pKN402A. The dUTPase production in dut mutant strains carrying this plasmid (pKK141) was only at about wild-type level after temperature shift. To test the hypothesis that the SacI cleavage used affects a control region for the dut gene, we recloned the dut fragment by transferring it from pKK141 into pHUB2 (Bernard et al., Gene 5 (1979) 59-76), a plasmid carrying the phage lambda pL promoter. A 3.6-kb EcoRI-BamHI fragment from pKK141, including the 1.5-kb SacI-BamHI segment from the dut region, was inserted downstream from the pL promoter. When this plasmid was present in a strain containing a thermosensitive lambda repressor gene, thermoinduction of dUTPase was negligible, apparently due to the presence of some termination signals between pL and dut. Therefore, we removed a 1.9-kb EcoRI-SacI fragment from the region between pL and the dut gene and replaced it with a 0.22-kb EcoRI-SacI fragment, obtained from the b2 region of lambda. Strains carrying such a shortened dut-pHUB2 derivative and a temperature-sensitive lambda repressor overproduced dUTPase very dramatically after heat induction. The final level reached was 300-400 times the wild-type level, corresponding to 10% of the total soluble protein. The information obtained, together with analysis of plasmid-directed polypeptide products described by Lundberg et al. (Gene 22 (1983) 127-131) shows that the SacI site is indeed on the promoter-proximal side of the dut gene.

A two-step mutant lacking two periplasmic enzymes, alkaline phosphatase and 5′-nucleotidase, was prepared. In extracts of this strain phosphatase activities towards several 5′-nucleotides could be detected. We have partially purified a dUMP phosphatase by streptomycin precipitation and DEAE-chromatography. This preparation has significant phosphatase activity towards three substrates, viz. dUMP, dTMP and UMP, but only traces of activity towards nine other tested nucleoside monophosphates. The three activities are probably due to a single enzyme, since they decrease in parallel on long time storage at 4°C and respond in parallel to stimulatory and inhibitory influences of different buffers and metal ions. The best buffer tested is glycylglycine buffer; Mg2+ is required but 25% activity can be obtained with Co2+ and Mn2+ and 6–8% activity with Fe2+ and Ni2+. Ca2+, Cu2+ or Zn2+ inhibit the phosphatase activities in the presence of Mg2+ or Co2+. The activities towards UMP and dUMP in the DEAE—chromatography eluted identically. The enzyme has the remarkably high apparent Km of 10-2 M with all three substrates.

The ribonucleotide reductase system of Escherichia coli B participates in the biosynthesis of DNA by reducing ribonucleoside diphosphates to the corresponding deoxyribonucleotides. The enzyme is regulated in a complicated way by allosteric modifiers. We now find that infection of E. coli with the bacteriophage T4 results in the appearance of a new ribonucleotide reductase system which shows a somewhat different pattern of regulation. Two new proteins, provisionally called fractions A and B, were purified from the extracts of infected bacteria. The reduction of ribonucleotides by these two fractions required the addition of either dithiothreitol or TPNH and E. coli thioredoxin reductase. Mutants of T4 which lacked fraction A activity were obtained. Fraction B may be a virus-induced thioredoxin.

The E2F transcription factors are key targets for the retinoblastoma protein, pRB. By inactivation of E2Fs, pRB prevents progression to the S phase. To test proliferative functions of E2F, we generated transgenic mice expressing human E2F-1 and/or human DP-1. When the hydroxymethyl glutaryl coenzyme A reductase promoter was used to express DP-1, overexpression occurred in a variety of tissues and did not confer phenotypic changes. In contrast, expression of E2F-1 from the same promoter was obtained only in testicles, in which E2F-1 overexpression caused atrophy and sterility through a process involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered testicular atrophy.