SPiRIT study protocol (Shoulder Pain: Randomised trial of Injectable Treatments): a randomised feasibility and pilot study of autologous protein solution (APS) vs corticosteroids for treating subacromial shoulder pain.

BACKGROUND: The management of subacromial shoulder pain represents a significant challenge and is typically managed through either physiotherapy, joint injection or surgical intervention. Recent surgical trials have questioned the efficacy and there is a need to improve the evidence base for the non-surgical management of this condition. The study aims to provide evidence of the feasibility of conducting a randomised controlled trial to compare the efficacy of autologous protein solution (APS) against the current standard of care, corticosteroid injection (CSI) for subacromial shoulder pain. Autologous protein solution (APS) is a blood-derived biological injection which has been shown to have anti-inflammatory effects. METHODS: A parallel-group two-arm randomised control trial will be conducted, comparing APS and CSI for shoulder pain. Fifty patients will be recruited. Feasibility will be assessed by examination of the conversion rate of eligible participants to the total number of participants recruited, whether it is possible to collect the appropriate outcome measures and the levels of retention/data compliance at follow-up dates. DISCUSSION: CSI is the mainstay of conservative management of subacromial shoulder pain. Trials and systematic reviews have reported differing conclusions, but the consensus view is that any benefits seen from CSI use are most likely to be short-term and there remains a significant number of patients who go on to have surgical intervention despite CSI. Biological injections, such as APS are being increasingly used, in the anticipation they may offer improved longer lasting outcomes for shoulder pain. However, the evidence to demonstrate the comparative efficacy of CSI versus APS does not currently exist. If feasible, a fully powered study will offer clarity to the treatment pathway of thousands of patients each year with subacromial pain. TRIAL REGISTRATION: The study is funded by the National Institute for Health Research-Research for Patient Benefit, NIHR 201473, Trial Registration Number (ISRCTN12536844: SPiRIT. Shoulder pain: randomised trial of injectable treatments-date of Registration 15/9/2021). Protocol Version V1.0_30Jul2021. IRAS Project ID: 294,982.

The Scottish Brain Health Service Model: Rationale and Scientific Basis for a National Care Pathway of Brain Health Services in Scotland.

In order to address the oft-cited societal, economic, and health and social care impacts of neurodegenerative diseases, such as Alzheimer's disease, we must move decisively from reactive to proactive clinical practice and to embed evidence-based brain health education throughout society. Most disease processes can be at least partially prevented, slowed, or reversed. We have long neglected to intervene in neurodegenerative disease processes, largely due to a misconception that their predominant symptom - cognitive decline - is a normal, age-related process, but also due to a lack of multi-disciplinary collaboration. We now understand that there are modifiable risk factors for neurodegenerative diseases, that successful management of common comorbidities (such as diabetes and hypertension) can reduce the incidence of neurodegenerative disease, and that disease processes begin (and, crucially, can be detected, reduced, and delayed, prevented, or treated) decades earlier in life than had previously been appreciated. Brain Health Scotland, established by Scottish Government and working in partnership with Alzheimer Scotland, propose far-reaching public health and clinical practice approaches to reduce neurodegenerative disease incidence. Focusing here on Brain Health Scotland's clinical offerings, we present the Scottish Model for Brain Health Services. To our knowledge, the Scottish Model for Brain Health, built on foundations of personalised risk profiling, targeted risk reduction and prevention, early disease detection, equity of access, and harnessing comprehensive data to assist in clinical decision-making, marks the first example of a nationwide approach to overhauling clinical, societal, and political approaches to the prevention, assessment, and treatment of neurodegenerative disease.

Probiotic guidelines and physician practice: a cross-sectional survey and overview of the literature.

Probiotic use by patients and physicians has dramatically increased over the last decade, although definitive evidence is often lacking for their use. We examined probiotic-prescribing practices among health care providers (HCP) at a tertiary medical centre and compared these practices to clinical guidelines. HCP at the Stanford Medical Center received a survey on probiotic prescribing practices including choice of probiotic and primary indications. A broad overview of the literature was performed. Among 2,331 HCP surveyed, 632 responded. Of the 582 of these who routinely prescribed medications, 61% had recommended probiotic foods or supplements to their patients. Women and gastroenterologists were more likely to prescribe probiotics (odds ratio (OR): 1.5, 95% confidence interval (CI): 1.0-2.1; OR: 3.9, 95% CI: 1.5-10.1, respectively). Among probiotic prescribers, 50% prescribed inconsistently or upon patient request, and 40% left probiotic choice to the patient. Common indications for probiotics, particularly Lactobacillus GG, were prevention and treatment of antibiotic-associated diarrhoea (79 and 66%, respectively). Probiotics were often prescribed for 'general bowel health' or at patient request (27 and 39% of responders, respectively). Most respondents (63%) thought an electronic medical record (EMR) pop-up would change probiotic prescribing patterns. However, a review of published guidelines and large trials found inconsistencies in probiotic indications, dosages and strain selection. Probiotic prescribing is common but lacks consistency, with choice of probiotic frequently left to the patient, even for indications with some strain-specific evidence. Implementation of EMR pop-ups/pocket guides may increase consistency in probiotic prescribing, although the lack of clear and consistent guidelines must first be addressed with large, well-designed clinical trials.

A survey of probiotic use practices among patients at a tertiary medical centre.

Probiotic use has skyrocketed in recent years. Little is known, however, about patient knowledge and practices regarding probiotic use, especially in the context of antibiotic use. An invitation to complete a short, anonymous, electronic survey was sent by email to 965 patients at a tertiary medical centre in California who had agreed to be contacted for participation in research studies. Questions were asked about both probiotic and antibiotic use in the prior three months. Of 333 survey respondents, 55% had recently used probiotics, including food products and/or supplements (90 and 60% of probiotic users, respectively). Women were more likely than men to have used probiotics (odds ratio (OR): 1.99; 95% confidence interval (CI): 1.2-3.4). Health care providers (HCP) had prescribed antibiotics to 79 (24%) respondents in the preceding three months. Among antibiotic users, 33% had initiated or changed probiotics at the time of antibiotic use, usually without a recommendation from their prescribing HCP (72%). Only 12% of those who took probiotics with antibiotics had received a specific recommendation from their HCP. Most patients chose to take probiotic mixtures (56%), with few selecting evidence-based strains, such as Lactobacillus rhamnosus GG (11%). Regular probiotic use among patients is common. Typically, these probiotics are not recommended by a HCP, even in conjunction with antibiotic prescriptions. While a growing body of evidence supports specific probiotic strains for the prevention of antibiotic-associated diarrhoea, patients are often not receiving a specific recommendation from their HCP and appear to be taking strains without guidance from supporting evidence.

A single amino acid substitution in the envelope protein of chimeric yellow fever-dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys.

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

Safety and efficacy of chimeric yellow Fever-dengue virus tetravalent vaccine formulations in nonhuman primates.

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.

Multimedia article. Laparoscopic Ladd's procedure in two adults: malrotation and the minimally invasive approach.

Malrotation is an intestinal rotation anomaly rarely diagnosed in adults. In the adult patient, obstructing peritoneal bands may lead to nausea and abdominal distention. Familiarity with this presentation as well as the aberrant anatomy associated with the unusual problem facilitates surgical treatment. While the minimally invasive approach requires meticulous dissection due to this abnormal anatomy, laparoscopic treatment does provide the advantages of short convalescence and low morbidity. This video briefly reviews embryologic intestinal development, rotational anomalies and two laparoscopic Ladd's procedures.

Laparoscopic cholecystectomy for triple gallbladder.

A very rare case involving true triplication of the gallbladder in a 38-year-old man with no other congenital abnormalities is reported. All three gallbladders had signs of chronic inflammatory disease and lithiasis. Acute cholecystitis and a double gallbladder were diagnosed preoperatively, but the diagnosis of a second accessory gallbladder was made only at the time of surgery. The patient underwent successful laparoscopic cholecystectomy. When a triplicate gallbladder is encountered, complete dissection of Calot's triangle and intraoperative cholangiographic evaluation should be performed to prevent damage to the biliary duct system. All three gallbladders should be removed to avoid unnecessary reoperation. All of this can be accomplished readily by laparoscopic surgery.

Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates.

Chimeric yellow fever (YF)-dengue (DEN) viruses (ChimeriVax-DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290-7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [ approximately 7 to 8 log(10), plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log(10) PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax-DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.

Laparoscopy and the internet. A surgeon survey.

BACKGROUND: The Internet has become an important new tool for the delivery and acquisition of medical information. METHODS: A 13-item questionnaire designed to collect information on the attitudes and practices of surgeons regarding the use of the Internet as a medical resource was posted on the World Wide Web and also sent via e-mail. RESULTS: Over a 2-month period, 459 surgeons were enrolled in this study. Most of the respondents were identified as male surgeons (96%) between the ages of 31 and 50 years (79.25%). They accessed the Internet mainly from their homes (67.10%) and offices (17%) using 56 Kbps (34.86%) and 33.6 Kbps (21.79%) modems. These participants indicated that they use the Internet to expand their knowledge of general surgery (78.87%), learn more about technologies related to the practice of surgery (74.51%), access the Medline medical database (73.20%), and locate other resources for academic purposes (68%). Approximately half of them said that they favored the use of robotic assist devices in the operating room (53%), and most supported the use of technology for telementoring purposes (78%). Almost 80% professed an interest in video streaming technology applied to surgical education. CONCLUSIONS: This study showed that the Internet is a useful and powerful real-time survey tool that can help us to assess the impact of the World Wide Web and related technologies on surgical education and practice. However, the respondents in this study belong to a biased group that is already familiar with the Internet and computer technology and thus may not be representative of the surgical community as a whole.

What happens to unplaced internship applicants in psychology?

Placement outcomes of students who did not secure internship offers on Uniform Notification Day 1997 were examined. An initial survey of unplaced students (n = 168) suggested mixed outcomes for students in obtaining sites after notification day, including negative emotional effects as a result of nonplacement. A follow-up survey completed by 77 students (43 who had not been placed during the original survey and 34 who had) suggested generally high satisfaction and optimism with internship and employment outlook despite their experiences with the internship application process. Accreditation concerns, emotional, time, and financial expenditures, and potential courses of action are discussed. Concrete suggestions for improving the selection system are provided.

An algorithm for automatic needle localization in ultrasound-guided breast biopsies.

An algorithm was developed in order to reduce operator dependence in ultrasound-guided breast biopsy, by automatically locating the needle in the ultrasound image, and displaying its location on the image for the user. Ultrasound images of a typical breast biopsy needle inserted in a tissue-mimicking agar were obtained to test the algorithm. The resulting images were examined by a group of observers who recorded the values of the angle, intercept and tip coordinates of the needle in the image, and inter- and intra-observer variability studies were performed on the results. The results of the algorithm segmentation were compared to the values recorded by the observers, and physical measurements recorded at the time the images were acquired. The algorithm segmentation was precise enough to successfully (when considering angle and tip segmentation) target 90% of tumors of 4.5 mm in diameter situated at the center of the image.

Intact mitochondrial electron transport function is essential for signalling by hydrogen peroxide in cardiac myocytes.

Oxidative stress has been proposed as a mediator of cardiac injury during ischemia and reperfusion. We examined the signalling events initiated by short-term exposure of cardiac myocytes to oxidative stress elicited by hydrogen peroxide. A potent stimulation of tyrosine phosphorylation was observed within 1 to 2 min exposure to 1 m m hydrogen peroxide. Within 5 min, the ERK mitogen-activated protein kinases (ERK MAPKs) were activated. This activation of ERK MAPKs was blocked by N-acetylcysteine (NAC), implicating a role for free radicals in the signalling events. NAC failed to inhibit ERK MAPK activation by the hypertrophic agent, phenylephrine, or hyperosmotic shock. Myxothiazol, an inhibitor of complex III of the mitochondrial electron transport chain, also inhibited ERK MAPK activation by hydrogen peroxide, but not by 12- O -tetradecanoylphorbol-13-acetate (TPA) or hyperosmotic shock. Myxothiazol completely inhibited the increase in tyrosine phosphorylated proteins observed with hydrogen peroxide treatment. A variety of inhibitors which act at different levels of the mitochondrial electron transport chain (rotenone, theonyltrifluoroacetone, antimycin A, cyanide) also inhibited activation of the ERK MAPKs by hydrogen peroxide but not TPA or hyperosmotic shock. These studies suggest a novel mechanism of regulation of the ERK MAPK pathway and oxidative stress signalling by hydrogen peroxide.

Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys.

ChimeriVax-JE is a live, attenuated recombinant virus prepared by replacing the genes encoding two structural proteins (prM and E) of yellow fever 17D virus with the corresponding genes of an attenuated strain of Japanese encephalitis virus (JE), SA14-14-2 (T. J. Chambers et al., J. Virol. 73:3095-3101, 1999). Since the prM and E proteins contain antigens conferring protective humoral and cellular immunity, the immune response to vaccination is directed principally at JE. The prM-E genome sequence of the ChimeriVax-JE in diploid fetal rhesus lung cells (FRhL, a substrate acceptable for human vaccines) was identical to that of JE SA14-14-2 vaccine and differed from sequences of virulent wild-type strains (SA14 and Nakayama) at six amino acid residues in the envelope gene (E107, E138, E176, E279, E315, and E439). ChimeriVax-JE was fully attenuated for weaned mice inoculated by the intracerebral (i.c.) route, whereas commercial yellow fever 17D vaccine (YF-Vax) caused lethal encephalitis with a 50% lethal dose of 1.67 log(10) PFU. Groups of four rhesus monkeys were inoculated by the subcutaneous route with 2.0, 3.0, 4.0, and 5. 0 log(10) PFU of ChimeriVax-JE. All 16 monkeys developed low viremias (mean peak viremia, 1.7 to 2.1 log(10) PFU/ml; mean duration, 1.8 to 2.3 days). Neutralizing antibodies appeared between days 6 and 10; by day 30, neutralizing antibody responses were similar across dose groups. Neutralizing antibody titers to the homologous (vaccine) strain were higher than to the heterologous wild-type JE strains. All immunized monkeys and sham-immunized controls were challenged i.c. on day 54 with 5.2 log(10) PFU of wild-type JE. None of the immunized monkeys developed viremia or illness and had mild residual brain lesions, whereas controls developed viremia, clinical encephalitis, and severe histopathologic lesions. Immunized monkeys developed significant (>/=4-fold) increases in serum and cerebrospinal fluid neutralizing antibodies after i.c. challenge. In a standardized test for neurovirulence, ChimeriVax-JE and YF-Vax were compared in groups of 10 monkeys inoculated i.c. and analyzed histopathologically on day 30. Lesion scores in brains and spinal cord were significantly higher for monkeys inoculated with YF-Vax. ChimeriVax-JE meets preclinical safety and efficacy requirements for a human vaccine; it appears safer than yellow fever 17D vaccine but has a similar profile of immunogenicity and protective efficacy.

Duration of postlaparoscopic pneumoperitoneum.

BACKGROUND: Patients who present with abdominal pain after recent laparoscopic surgery present a diagnostic dilemma when pneumoperitoneum is present. Previous studies do not define the duration of postlaparoscopic pneumoperitoneum. In this study, we attempted to define the duration of laparoscopic pneumoperitoneum and to identify factors which affect resolution time. METHODS: We followed 57 patients who underwent laparoscopic cholecystectomy (34), inguinal herniorraphy (20), or appendectomy (three). Serial abdominal films were taken until all residual gas was resolved. RESULTS: Thirty patients resolved their pneumoperitoneum within 24 h; 16 patients resolved between 24 h and 3 days; nine patients resolved between 3 and 7 days; two patients resolved between 7 and 9 days. Mean resolution time for all patients was 2.6 +/- 2.1 days. There was no apparent difference in resolution time between the three types of procedures; however, the sample size may be insufficient. Duration of the pneumoperitoneum did not correlate with gender, age, weight, initial volume of CO2 used, length of time for the procedure, or postoperative complications. Sixteen patients had bile spillage during cholecystectomy which significantly reduced the duration of postoperative pneumoperitoneum (p < 0.008), resulting in a mean resolution time of 1.3 +/- 0.9 days. While 14 patients reported postoperative shoulder pain, no correlation was found between the presence or duration of shoulder pain and the extent or duration of pneumoperitoneum. CONCLUSIONS: We conclude that the residual pneumoperitoneum following laparoscopic surgery resolves within 3 days in 81% of patients and within 7 days in 96% of patients. The resolution time was significantly less in patients sustaining intraoperative bile spillage during cholecystectomy. There was no correlation found between postoperative shoulder pain and the presence or duration of the pneumoperitoneum.

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2'-O-Me nucleotides, 1-2 other 2'-modified nucleotides at positions U4 and U7, and a 3'-3'-linked nucleotide "cap." Eight 2'-modified uridine residues were introduced at positions U4 and U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 substitutions at U4 and U7, or 2'-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only approximately 0.1 min. The addition of a 3'-3'-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to > 260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

Assessment of training in psychosexual medicine.

The Institute of Psychosexual Medicine offers training in the treatment of psychosexual problems to medically qualified doctors. Training takes place in fortnightly seminars in which trainees present and discuss real cases. Assessment of cases presented at the beginning and end of the six term basic training showed appreciable improvement in doctors' abilities. The proportion of doctors meeting each of the 14 predetermined clinical objectives rose. Factors which affected the amount of improvement were the initial score, the number of cases presented at the seminars, the occupation of the leader, and the duration of training. Accreditation by the Institute of Psychosexual Medicine was shown to be an appropriate outcome measure for the achievement of the required standards for practising psychosexual medicine.

Mobilizing affect: a possible effect of day hospital treatment for chronic psychiatric patients.

A study of 82 Psychiatric Day Hospital patients was undertaken to identify the program's specific effects on individuals with longer standing (i.e., chronic) psychiatric disability. Sociodemographic information and self-ratings, staff ratings and significant-other ratings were used to identify changes in functioning during the 3-week treatment as well as during the period 3 months after treatment. Findings suggest (1) that the Day Hospital patients were as seriously psychiatrically impaired as psychiatric inpatients, (2) that, as a group, they demonstrated a significant improvement in symptoms and functioning, and (3) that the more chronic patients displayed a distinctive pattern of decreased hostility and increased anxiety over the course of treatment. Findings are discussed in relation to the proposition that mobilizing the chronic patient's affect is an important factor in reengaging the therapeutic process.

Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection.

Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNAse H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.