Engineering herpes simplex virus vectors for human gene therapy.
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Animals↗
Biomedical subjects
R Ramakrishnan
Publications and source records attributed to R Ramakrishnan.
The initial complication rate of phacoemulsification in India.
PURPOSE: This study was designed to investigate the feasibility of teaching experienced surgeons to perform phacoemulsification in India, a cataract-endemic area. Complications occurring during surgery and the first postoperative day were documented and evaluated. METHODS: During a 1-month period, at the Aravind Eye Hospital in Madurai, India, the first 100 consecutive cataract operations performed by each of three experienced surgeons (a total of 300 cases), using phacoemulsification were prospectively evaluated. Multiple logistic regression was used to identify factors associated with intraoperative and postoperative complications. RESULTS: The mean age of patients was 57.4+/-9.3 years. The median best corrected preoperative visual acuity was 20/80. Mean surgical and phacoemulsification times were 15.8+/-3.7 minutes and 2.2+/-1.5 minutes, respectively. Complications occurred in 65 (21.7%) eyes. The most common was a rent in the posterior capsule, occurring in 40 (13.3%) eyes. There were significant variations in complication rate and in surgical time among the surgeons. The risk of experiencing a complication decreased as the number of phacoemulsifications performed increased. An increased risk of complications was associated with worse preoperative visual acuity and increasing patient age. CONCLUSIONS: With each successive case, the chances of experiencing a complication decreased 1%. Acceptable results were obtained within 1 month of performing the first phacoemulsification.
Adult↗
Replication-defective herpes simplex virus vectors for gene transfer in vivo.
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Animals↗
Combined hyperlipidemia in transgenic mice overexpressing human apolipoprotein Cl.
We have generated transgenic mice over-expressing human apolipoprotein CI (apo CI) using the native gene joined to the downstream 154-bp liver-specific enhancer that we defined for apo E. Human apo CI (HuCI)-transgenic mice showed elevation of plasma triglycerides (mg/dl) compared to controls in both the fasted (211 +/- 81 vs 123 +/- 52, P = 0.0001) and fed (265 +/- 105 vs 146 +/- 68, P < 0.0001) states. Unlike the human apo CII (HuCII)- and apo CIII (HuCIII)-transgenic mouse models of hypertriglyceridemia, plasma cholesterol was disproportionately elevated (95 +/- 23 vs 73 +/- 23, P = 0.002, fasted and 90 +/- 24 vs 61 +/- 14, P < 0.0001, fed). Lipoprotein fractionation showed increased VLDL and IDL + LDL with an increased cholesterol/triglyceride ratio (0.114 vs 0.065, P = 0.02, in VLDL). The VLDL apo E/apo B ratio was decreased 3.4-fold (P = 0.05) and apo CII and apo CIII decreased in proportion to apo E. Triglyceride and apo B production rates were normal, but clearance rates of VLDL triglycerides and postlipolysis lipoprotein "remnants" were significantly slowed. Plasma apo B was significantly elevated. Unlike HuCII- and HuCIII-transgenic mice, VLDL from HuCI transgenic mice bound heparin-Sepharose, a model for cell-surface glycosaminoglycans, normally. In summary, apo CI overexpression is associated with decreased particulate uptake of apo B-containing lipoproteins, leading to increased levels of several potentially atherogenic species, including cholesterol-enriched VLDL, IDL, and LDL.
Animals↗
Physicians' metered dose inhaler technique after a single teaching session.
BACKGROUND: It is known that many housestaff physicians are unable to demonstrate perfect metered dose inhaler (MDI) technique. OBJECTIVE: This study assessed whether a single teaching session for house staff physicians would significantly improve their MDI technique. METHODS: Thirty-eight pediatric house staff physicians were asked to demonstrate MDI technique with a placebo MDI. The physicians were evaluated on the following seven steps: (1) shaking the MDI and removing the cap, (2) exhaling prior to MDI use, (3) holding the MDI upright, (4) proper timing of actuation, (5) a slow inspiratory effort, (6) one MDI actuation per breath, and (7) holding the breath > or = 5 seconds. A 20-minute teaching session and demonstration of proper MDI technique was then given. At the end of this session all residents were eventually able to demonstrate proper technique. Two months following this educational session the same house staff physicians were re-evaluated on their MDI technique. RESULTS: Initially, ten participants (26%) demonstrated perfect technique. Two months postinstruction the same number of physicians (ten) demonstrated perfect technique. Only six physicians demonstrated perfect technique at both evaluations. Three of the seven steps showed enough change from the first evaluation to the second to permit statistical analysis. Step 4 (timing of actuation) had 11 Physicians' performances improve while three worsened (P=.03). Step 5 (a slow inspiratory effort) had nine physicians' performances improve while three worsened (P=.073). For step 7 (holding the breath > or = 5 seconds), 11 physicians improved while 2 worsened (P=.006). Comparing global performance, there were 17 physicians that improved, 8 that worsened, and 13 with no change (P=.054). CONCLUSIONS: This study confirmed that many housestaff physicians do not demonstrate optimal MDI technique. While one educational session may somewhat improve their future performance, it is not sufficient to guarantee perfect technique. This suggests that repeated education needs to be given to housestaff physicians.
Administration, Inhalation↗
cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo.
Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.
Animals↗
Detection of herpes simplex virus type 1 latency-associated transcript expression in trigeminal ganglia by in situ reverse transcriptase PCR.
One of the defining characteristics of herpes simplex virus type 1 (HSV-1) infection is the ability of the virus to establish a lifelong latent state in neurons. We previously demonstrated (R. Ramakrishnan, A.J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994) by in situ PCR that many more neurons contain viral genomes than are detected by in situ hybridization for HSV latency-associated transcripts (LATs). To determine whether all cells which contain genomes express LATs, we examined trigeminal ganglia for LATs 1 and 8 weeks after corneal scarification with ribonucleotide reductase-deficient HSV-1 by in situ reverse transcriptase PCR. The number of LAT-positive cells detected by in situ reverse transcriptase was substantially greater than the number of cells positive by in situ hybridization and appeared to be similar to the number of cells containing HSV genomes by in situ PCR and the number of ganglionic neurons that project to the cornea as detected by retrograde labeling with Fluorogold. These results demonstrate LAT expression in many neurons containing HSV-1 genomes.
Animals↗
Effect of vitamin A supplementation to mother and infant on morbidity in infancy.
OBJECTIVES: To assess the impact of Vitamin A supplementation to the mother soon after delivery and to the infant at six months on morbidity in infancy. DESIGN: Randomized double blind placebo controlled field trial. SETTING: 51 villages in two contiguous Primary Health Centers in Villupuram Health Unit District of Tamil Nadu, South India. SUBJECTS: 909 newly delivered mother-and-infant pairs. INTERVENTIONS: Both mother and infant received Vitamin A (300,000 IU for mothers and 200,000 IU for children) in 311 instances (AA); mother received Vitamin A but infant received Placebo in 301 instances (AP); and both mother and infant received Placebo in the remaining 297 instances (PP). MAIN OUTCOME MEASURES: Incidence of diarrhea and Acute Respiratory Infection (ARI); distributions of infants by frequency of episodes and number of infected days. RESULTS: 233 in the AA Group and 228 each in the AP and PP Groups were followed up regularly. The incidence of diarrhea in these infants was 97.4%, 96.9% and 94.7% in the three groups, mean number of diarrheal episodes was 4.4, 4.6 and 4.2 and median number of days in infancy with diarrhea was 26, 26 and 22 days, respectively. For ARI, the incidences were 96.6%, 95.6% and 96.1%, means were 4.8, 5.1 and 4.8 episodes, and the medians were 32, 34 and 34 days, respectively. CONCLUSIONS: Prophylactic administration of mega doses of Vitamin A to the mother soon after delivery and to the infant at six months do not have any beneficial impact on the incidence of diarrhea and ARI in infancy.
Adult↗
High-volume intraocular lens surgery in a rural eye camp in India.
BACKGROUND AND OBJECTIVE: The appropriate surgical treatment for the enormous number of patients in developing nations who are blind due to cataract is a hotly debated issue. The authors objective is to demonstrate that modern surgical techniques (extracapsular cataract extraction and intraocular lens implantation, phacoemulsification and intraocular lens implantation) can be performed in a high-volume, cost effective manner, even in temporary settings. The authors believe that the approach to cataract blindness is not simple intracapsular cataract extraction, but rather the challenge of (1) training all ophthalmic personnel in modern techniques (microsurgery, biometry), (2) training managers in higher levels of organizational skill, and (3) doing these things in the face of limited resources. PATIENTS AND METHODS: A total of 1298 surgeries were performed in a public eye camp in Ganeshpuri, India (50 miles north of Bombay). Of these, 1214 (93.5%) of the patients received intraocular lens (IOL) implants. Ninety-three percent (1032/1108) of the patients who underwent extracapsular cataract extraction (ECCE) and IOL implantation and 89% (83/93) of the patients who underwent phacoemulsification and IOL implantation returned for follow-up. RESULTS: Postoperatively, 48% (498/1032) of the patients who underwent ECCE and IOL implantation achieved corrected vision of 6/12 or better and 65% (671/1032) attained corrected vision of 6/18 or better. Of the patients who underwent phacoemulsification and IOL implantation, 59 of 83 (71%) attained vision of 6/12 or better with correction and 68 of 83 (79%) achieved vision of 6/18 or better with correction. These results are almost identical to those obtained by the authors in their Ganeshpuri 1991 camp. Surgical complication rates were comparable to those reported in hospital-based studies. CONCLUSION: For this type of camp to operate efficiently, there must be standardization of skills among ophthalmic personnel, costs must be contained, and the organizational skills necessary to ensure smooth functioning of the camp must exist. However, on the basis of their data, these authors believe that with suitable organizational and surgical facilities, IOL implantation can be successfully performed in high-volume surgical eye camps.
Blindness↗
Lens induced glaucomas--visual results and risk factors for final visual acuity.
Lens induced glaucomas are a common occurrence in India. An attempt was made to understand the clinical modes of presentation and post operative visual results in 93 patients with lens induced glaucoma, 49 phacomorphic and 44 phacolytic, attending our institute during 1994. All these patients were subjected to a planned extracapsular cataract extraction. Forty four percent had a posterior chamber intraocular lens implantation following surgery. Fifty seven percent eyes with phacomorphic glaucoma and 61% with phacolytic glaucoma recovered visual acuity of 6/12 or better. There was no significant difference in the final visual acuity between those patients who had an intraocular lens implanted and those who did not (P = 0.18). Univariate analysis was performed for selected risk factors such as age, sex and duration of glaucomatous process as predictors of final visual acuity and odds ratios with 95% confidence intervals were calculated. Patients with age more than 60 years (OR = 2.7, 95% CI = 1.04-6.93) and in whom the glaucoma was present for more than 5 days (OR = 3.1, 95% CI = 1.21-8.13) had a significantly higher risk of poor visual outcome post-operatively.
Adult↗
Brain dopamine in experimental diabetes.
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Animals↗
Plasma delivery of retinoic acid to tissues in the rat.
All-trans-retinoic acid (RA) activates ligand-dependent transcription factors that regulate retinoid-responsive gene expression. It is assumed that all-trans-RA is formed within cells through in situ oxidation of retinol derived from the circulation. However, the circulation contains low levels of all-trans-RA (approximately 0.2-0.7% of that of plasma retinol). Our studies investigated the extent to which plasma all-trans-RA contributes to tissue pools of this retinoid and explored factors responsible for regulating its uptake by tissues and cells. Rats were continuously infused, to steady state, with all-trans-[3H]RA. From measures of specific activities of all-trans-[3H]RA at steady state, we determined that the preponderance of all-trans-RA in brain and liver was derived from the circulation. For six other tissues, approximately 10-30% of the retinoid was derived from the circulation, but pancreas and testis derived very little from the circulating pool. In other studies, we showed that retinoid nutritional status influences clearance of a bolus dose of all-trans-RA and that neither the rate of cellular all-trans-RA uptake nor its intracellular half-life is influenced by cellular lipid levels. Taken together, our data indicate that plasma all-trans-RA contributes to tissue pools of this retinoid and that specific and physiologically responsive cellular processes mediate its uptake.
Adipocytes↗
Norepinephrine and epinephrine levels in the brain of alloxan diabetic rats.
Thirty days after induction of experimental diabetes the brain catecholamines namely, norepinephrine (NE) and epinephrine (E) were studied in discrete brain regions (striatum, hippocampus, hypothalamus, midbrain, pons and medulla, cerebellum and cerebral cortex) in control, alloxan-diabetic untreated and insulin-treated diabetic rats. E showed significant increase in striatum, hippocampus and hypothalamus, whereas NE was increased in hypothalamus, and decreased in pons and medulla significantly in untreated diabetic rats. These effects were not seen in the insulin-treated diabetic rats.
Animals↗
Increases in dietary cholesterol are associated with modest increases in both LDL and HDL cholesterol in healthy young women.
We studied the effects of dietary cholesterol intake on lipid and lipoprotein levels in healthy young women (n = 13) who were otherwise eating an American Heart Association (AHA) diet. The study used a randomized, three-way crossover design to determine the effects of 0, 1, or 3 eggs added per day (dietary cholesterol range, 108 to 667 mg/d). Each of the three diets was eaten for 8 weeks, with a washout period between diets. Three fasting blood samples were obtained during the last 3 weeks of each diet period to observe changes in fasting plasma lipid levels associated with the menstrual cycle. We also obtained blood just before and 4 and 8 hours after the subjects ingested a standard high-fat formula. During the menstrual cycle, total cholesterol and LDL cholesterol levels fell by 0.051 mmol/L (1.99 mg/dL) and 0.064 mmol/L (2.48 mg/dL) per week, respectively. HDL cholesterol concentrations increased by 0.060 mmol/L (2.3 mg/dL) per week during the first half of the cycle and then fell by 0.050 mmol/L (1.94 mg/dL) per week during the second half. Therefore, all statistical analyses were performed on values adjusted to midcycle. Total fasting cholesterol concentrations increased by 0.073 mmol/L (2.81 mg/dL) per 100 mg dietary cholesterol added to the diet per day (P = .001). LDL cholesterol increased by 0.054 mmol/L (2.08 mg/dL) per 100 mg/d dietary cholesterol (P = .003); this accounted for about 75% of the rise in total cholesterol. HDL cholesterol concentrations increased by 0.015 mmol/L (0.57 mg/dL) per 100 mg/d dietary cholesterol (P < .04). There was a wide range of responses among the women. Plasma apoB levels increased significantly, 0.93 mg/dL per 100 mg/d dietary cholesterol (P = .025), whereas apoA-I levels tended to rise (1.35 mg/dL per 100 mg/d, P = .056). Increases in dietary cholesterol did not produce any observable effects on fasting plasma cholesteryl ester transfer protein levels and had no effect on the response to a standard high-fat formula. Although menstrual-cycle changes in plasma total, LDL, and HDL cholesterol levels were observed, the effects of the diets were similar in the follicular and luteal phases of the menstrual cycle. Additionally, despite changes associated with the menstrual cycle, within-subject variation in plasma total cholesterol was actually smaller in this study than in our study of young men.
Adult↗
Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.
Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences.
Adipose Tissue↗
Randomised controlled field trial of two antenatal care packages in rural south India.
A randomised community intervention trial was undertaken in 12 subcentres in Karur health unit district, Tamil Nadu, to compare the efficacy of two antenatal care packages. A newly recommended 'high-risk' strategy package and a uniform package recommended by the Tamil Nadu Government were each implemented in 4 randomly selected subcentres by the study team, and the latter was also delivered by routine health services in the 4 remaining subcentres. Analyses were based on 294 pregnant women on the high-risk package (HR), 242 on the Tamil Nadu Government (TNG) package and 335 women in the control series. The HR package reduced the differences between the high-risk women and the others in mean haemoglobin and the percentages with preventable neonatal morbidity and low birth weight, and consequently the overall outcome was better in the HR series than in the TNG series. Finally, the results with the TNG package were better when it was implemented by the study team than by the routine health services, in terms of preventable maternal morbidity and preventable perinatal morbidity, but there was no impact on birth weight.
Birth Weight↗
Human plasma vitamin E kinetics demonstrate rapid recycling of plasma RRR-alpha-tocopherol.
A kinetic model of vitamin E transport in humans is described using data from our studies with deuterium-labeled stereoisomers of alpha-tocopherol (RRR- and SRR-). In normal subjects, both alpha-tocopherols are present at similar concentrations in chylomicrons, but by 24 hr, RRR-alpha-tocopherol is at higher plasma concentrations because RRR-alpha-tocopherol is preferentially incorporated into very low density lipoproteins, which are then secreted into plasma. In three nondiscriminator patients with familial isolated vitamin E deficiency, the fractional disappearance rates (mean +/- SD) of deuterium-labeled RRR- and SRR-alpha-tocopherols in plasma were 1.4 +/- 0.6 and 1.3 +/- 0.3 pools per day, respectively (difference, 0.1 +/- 0.3). In these patients, plasma concentrations of both RRR- and SRR-alpha-tocopherols decreased similarly to SRR-alpha-tocopherol in controls. In six controls, fractional disappearance rates of deuterium-labeled RRR-alpha-tocopherol (0.4 +/- 0.1 pool per day) were significantly (P < 0.01) slower than for SRR- (1.2 +/- 0.6). The differences (0.8 +/- 0.6 pool per day) between these two rates in controls estimate the rate at which RRR-alpha-tocopherol, which had left the plasma, was returned to the plasma. Although plasma labeled RRR-alpha-tocopherol concentrations in controls appear to change slowly, these data show that both RRR- and SRR-alpha-tocopherols leave the plasma rapidly, but only RRR-alpha-tocopherol is returned to the plasma, likely in nascent very low density lipoproteins. This recycling of RRR-alpha-tocopherol accounts for nearly 1 pool of alpha-tocopherol per day.
Administration, Oral↗
The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.
Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane.
Amino Acid Sequence↗