Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

Pulmonary mechanics by spectral analysis of forced random noise.

The magnitude (Zrs) and phase angle (thetars) of the total respiratory impedance (Zrs), from 3 to 45 Hz, were rapidly obtained by a modification of the forced oscillation method, in which a random noise pressure wave is imposed on the respiratory system at the mouth and compared to the induced random flow using Fourier and spectral analysis. No significant amplitude or phase errors were introduced by the instrumentation. 10 normals, 5 smokers, and 5 patients with chronic obstructive lung disease (COPD) were studied. Measurements of Zrs were corrected for the parallel shunt impedance of the mouth, which was independently measured during a Valsalva maneuver, and from which the mechanical properties of the mouth were derived. There were small differences in Zrs between normals and smokers but both behaved approximately like a second-order system with thetars = 0 degree in the range of 5--9 Hz, and thetars in the range of +40 degrees at 20 Hz and +60 degrees at 40 Hz. In COPD, thetars remained more negative (compared to normals and smokers) at all frequencies and crossed 0 between 15 and 29 Hz. Changes in Zrs, similar in those in COPD, were also observed at low lung volumes in normals. These changes, the effects of a bronchodilator in COPD, and deviations of Zrs from second-order behavior in normals, can best be explained by a two-compartment parallel model, in which time-constant discrepancies between the lung parenchyma and compliant airway keep compliant greater than inertial reactance, resulting in a more negative phase angle as frequency is increased.

Predictors of success and major complications for primary percutaneous transluminal coronary angioplasty in acute myocardial infarction. An analysis of the 1990 to 1994 Society for Cardiac Angiography and Interventions registries.

OBJECTIVES: The purpose of this study was to determine predictors of successful coronary angioplasty for acute myocardial infarction (MI) and associated predictors of the major complications of in-hospital mortality and emergency coronary artery bypass graft surgery. BACKGROUND: Primary angioplasty is being increasingly used to treat acute MI, but factors affecting the success and major complications have not been well studied. Forty laboratories have been contributing clinical and procedural data to the Society of Cardiac Angiography and Interventions (SCA&I) on primary angioplasty for acute MI. METHODS: Univariable and stepwise multivariable logistic regression analysis of clinical and procedural variables was used to calculate predictors of success and major complications. RESULTS: There were 4,366 primary angioplasty procedures reported from 1990 through 1994, with an overall success rate of 91.5%, an in-hospital mortality rate of 2.5% and a rate of emergency surgery of 4.3%. Higher laboratory primary angioplasty volume and lower age were predictive of success. An intraaortic balloon pump in place, cardiogenic shock and a moribund condition had negative predictive effects. Unsuccessful angioplasty, cardiogenic shock or a moribund state were predictive of in-hospital death. Unsuccessful angioplasty, the absence of a history of hypertension and the absence of congestive heart failure were predictive of emergency surgery. CONCLUSIONS: The rates of success and major complications in the SCA&I Registry are similar to other series. Predictors of success and major complications can be assessed and may be useful for risk stratifying candidates for primary angioplasty in acute MI.

Assessment of the inotropic and vasodilator effects of amrinone versus isoproterenol.

The hemodynamic effects of graded-dose infusions of amrinone (maximal dose 30 micrograms/kg/min) (10 patients) and isoproterenol (maximum dose 4 micrograms/min) (11 patients) were assessed in patients with a range of left ventricular (LV) function. LV ejection fraction ranged from 0.13 to 0.77 (mean +/- standard deviation 0.47 +/- 0.23) among the patients who received amrinone and from 0.24 to 0.77 (mean 0.52 +/- 0.18) among those who received isoproterenol. Peak-dose amrinone produced a reduction in LV filling pressure (from 15 +/- 10 to 10 +/- 7 mm Hg, p less than 0.001), but no significant change in heart rate, cardiac output, mean aortic pressure, total systemic vascular resistance (TSVR) or LV dP/dt max. In contrast, peak-dose isoproterenol produced a similar reduction in LV filling pressure (from 17 +/- 12 to 13 +/- 13 mm Hg, p less than 0.05), but also caused increases in heart rate, cardiac output and LV dP/dt max and decreases in mean aortic pressure and TSVR (p less than 0.001). The absolute change in cardiac output and stroke volume correlated closely with the change in TSVR in response to amrinone (r = -0.90, p less than 0.001 and r = -0.84, p = 0.002, respectively), but not in response to isoproterenol. Although isoproterenol produced a marked increase in cardiac output and LV dP/dt max (not explained by heart rate changes alone) in all patients, amrinone produced an increase in cardiac output only in those with markedly elevated LV filling pressures (who had a reduction in TSVR), and an increase in LV dP/dt in a minority.

Hemodynamic effects of anti-G suit inflation in a 1-G environment.

This study evaluated effects of various anti-G inflation pressures on cardiac volumes and the relationship of these volume changes to mean arterial pressure changes. Ventricular volumes were calculated using two-dimensional echocardiography. An anti-G suit was inflated to 2, 4, and 6 psi in the standing and supine positions for 10 male subjects. In the supine position, mean arterial pressure increased from base line for all three inflation pressures (P = 0.05). The end-diastolic volume increased after 2-psi inflation (P = 0.03). Cardiac output or stroke volume did not change. After standing, mean arterial pressure (P = 0.002), end-diastolic volume (P = 0.002), and stroke volume (P = 0.05) fell after suit deflation. Peripheral vascular resistance fell in the 2- and 4-psi inflation profiles. In the standing protocol, mean arterial pressure, end-diastolic volume, stroke volume, and cardiac output rose with all three inflation pressures (P less than 0.05). After reclining, heart rate increased (P = 0.02) and mean arterial pressure fell (P less than 0.05) in the 4- and 6-psi inflation profiles after suit deflation. Increases in mean arterial pressure are caused by increases in cardiac preload and cardiac output after inflation of the anti-G suit while subjects were standing. Increased cardiac preload was not consistently seen after inflation while subjects were supine. Changes in end-diastolic volume and mean arterial pressure were dependent on the pressure used to inflate the anti-G suit.

Novastan anticoagulation during renal stent implant in a patient with heparin-induced thrombocytopenia.

We describe a case of a 70-year-old male who underwent coronary artery bypass surgery which was complicated by multiple thrombotic events associated with HIT. The thrombotic events were treated with intravenous argatroban (Novastan). During the hospitalization the patient was found to require percutaneous bilateral renal artery revascularization for acute renal failure. The revascularization procedure was successfully accomplished with a high dose argatroban regimen. We present our report of a successful anticoagulation strategy during a peripheral intervention in a patient with HIT and the laboratory data which support this strategy.

Failure of ancrod in the treatment of heparin-induced arterial thrombosis.

The morbidity and mortality associated with heparin-induced thrombosis remain high despite numerous empirical therapies. Ancrod has been used successfully for prophylaxis against development of thrombosis in patients with heparin induced platelet aggregation who require brief reexposure to heparin, but its success in patients who have developed the thrombosis syndrome is not well defined. The authors present a case of failure of ancrod treatment in a patient with heparin-induced thrombosis.

Technique of apical left ventricular puncture revisited: a case report of double-valve prosthesis evaluation.

The presence of both mitral and aortic mechanical prostheses often make access to the left ventricle difficult for hemodynamic evaluation of valve gradients and for performing angiography. We present a case where both the transseptal and direct apical ventricular puncture techniques are utilized for assessing prosthetic valve function. The use of a new over the wire technique for left ventricular access using a flexible 4 French pediatric multipurpose catheter is described.

A randomized trial of the low-molecular-weight heparin certoparin to prevent restenosis following coronary angioplasty.

OBJECTIVES: The objectives of this study were to evaluate the effectiveness and safety of the low-molecular-weight heparin (LMWH) certoparin in preventing restenosis following balloon coronary angioplasty. BACKGROUND: Restenosis following coronary angioplasty continues to limit the long-term efficacy of this procedure. Animal studies have indicated a potential role for LMWH in reducing restenosis by limiting smooth muscle proliferation. METHODS: This study tested the effects of certoparin, self-administered for 3 months, in reducing restenosis following balloon coronary angioplasty. One hundred and eighteen patients with 158 lesions treated with angioplasty were enrolled in this randomized, placebo-controlled trial. One hundred and two patients completed the study. The endpoint was relative loss measured with quantitative coronary angiography. RESULTS: The relative loss for placebo was 0.19 +/- 0.23 compared to 0.14 +/- 0.21 for LMWH (p = NS). The minimum lumen diameter (MLD) was 1.47 +/- 0.66 for placebo and 1.40 +/- 0.57 for the LMWH (p = NS). There was a reduction (31% for LMWH; 49% for placebo PSDP) in the percent of patients having binary restenosis (MLD < 50% of reference diameter). At the end of the study 77% of the placebo patients and 76% of the LMWH group were asymptomatic (p = NS). There was a low rate of bleeding complications and these were minor. Bone density scans showed that there was no significant occurrence of osteoporosis with 3 months of LMWH. CONCLUSIONS: Administration of certoparin for 3 months is safe, but appears ineffective in reducing post-PTCA restenosis.

Predictors of successful PTCA using coronary perfusion balloon catheters.

The purpose of this retrospective study was to define clinical, angiographic and procedural predictors of successful PTCA using perfusion balloon catheters (PBC). Age, gender, diabetes, clinical state (stable or unstable angina), coronary vessel, AHA/ACC lesion type, lesion contour, pre-procedural thrombus, percent stenosis, lesion length, balloon size, maximum PBC pressure, and maximum inflation time were analyzed for 207 lesions in 193 successive patients. Unsuccessful results occurred more frequently in patients with unstable angina, pre-PTCA thrombus, and those treated with smaller balloon catheter diameter. Logistical regression analysis identified larger balloon size (odds ratio [OR] = 0.447 [95% confidence interval 0.203, 0.986], p < .05); and absence of thrombus (OR = 2.217 [95% confidence interval 1.066, 4.610], p < .05) as predictors of success. This study suggests that small vessel size, approximated by balloon size selection, and the presence of pre-PTCA thrombus reduces the likelihood of success, especially in the setting of unstable angina. In these cases other percutaneous interventions may be warranted.

Assessment of outcome of bifurcation lesions and non-bifurcation lesions treated in the CAVEAT trial. CAVEAT investigator group.

Registry data suggests that bifurcation lesions are associated with reduced success during percutaneous revascularization. We studied 1012 CAVEAT patients to compare procedural outcomes in patients with and without bifurcation lesions whose target vessel was treated with either atherectomy or angioplasty. Bifurcation lesions have increased angiographic complexity and interventions on them are associated with lower acute procedural success rates compared to non-bifurcation lesions. Subgroup analysis suggests that atherectomy treatment of bifurcation lesions improves acute procedural success rates and lowers restenosis rates compared to angioplasty treatment of bifurcation lesions but atherectomy of bifurcation lesions is associated with higher acute complication rates than angioplasty of bifurcation lesions.

Improved hemodynamic, angiographic and functional results after renal artery stenting.

Eighteen patients with severe renal artery atherosclerosis underwent conventional percutaneous transluminal renal angioplasty (PTRA) followed immediately by implantation of an endovascular stent. Hemodynamic measurements showed a baseline trans-stenotic pressure gradient of 78.3 mmHg that was reduced to 14.8 mmHg after PTRA. The post PTRA trans-stenotic pressure gradient was further reduced to 0.86 mmHg after stent placement. The average baseline diameter stenosis of 81.3% was reduced to 43.7% after PTRA and 6.1% after stent placement. Six month angiographic follow-up revealed restenosis in 6/16 patients. In patients treated for chronic renal insufficiency without restenosis the 6 month creatinine was 1.46 mg/dl compared to a pre-procedure creatinine of 2.4 mg/dl. Therefore those patients with renal insufficiency and renal artery stenosis who had long term patency after successful stent implantation showed significant improvement in renal function at six months. Stent implantation also significantly improved acute hemodynamic results and acute angiographic results compared to conventional renal artery angioplasty.

Absence of respiratory sinus arrhythmia: a manifestation of the sick sinus syndrome.

The failure of changes in autonomic drive to affect heart rate is a common finding in patients with the sick sinus syndrome (SSS). The normal respiratory sinus arrhythmia is vagally mediated and should therefore be attenuated in SSS. This hypothesis was tested in a series of 32 symptomatic patients with SSS, mean age 68 years, and an age-matched control group of 75 normal subjects. The degree of sinus arrhythmia present in routine 12-lead electrocardiograms was quantitated as (1) the coefficient of variation (CV) of the R-R interval, and (2) the maximal R-R interval difference (delta R-Rmax). Mean CV was 2.7% in SSS compared to 3.8% in the control group (p less than 0.01). Mean delta R-Rmax was 72 ms in SSS and 140 ms in controls (p less than 0.01). Sixteen of 32 patients (50%) compared to only 4/75 (5%) control subjects had delta R-Rmax of less than 80 ms (p less than 0.01). Thus, SSS is frequently associated with a significant attenuation of sinus arrhythmia and the simple measurement of delta R-Rmax in standard ECG recordings provides useful diagnostic information.

Effect of ischemia on ventricular contractility in patients with coronary artery disease.

The constant of elastance (E-MAX) is determined by measuring continuous left ventricular pressure and volume changes during the cardiac cycle. To evaluate the effect of myocardial ischemia on contractility, E-MAX was measured from serial pressure volume loops at baseline, with atrial pacing-induced ischemia, and after the administration of intravenous nitroglycerin with repeat pacing. Ten patients undergoing cardiac catheterization for presumed coronary artery disease were evaluated by this method. The severity of the coronary artery disease was graded angiographically by using the Gensini coronary score. In 8 of the 10 patients there was a significant decrease in E-MAX during atrial pacing (P less than .05). With the administration of nitroglycerin there was an attenuation of the ischemic effect previously noted with pacing. In 3 of the 10 patients administration of nitroglycerin produced a further decrease in E-MAX with ischemia. All 3 patients had a significantly lower Gensini coronary score with a well-developed, visible coronary collateral network. The authors' results indicate that continuous pressure volume loop analysis is possible using the nuclear stethoscope. Significant ischemic changes are seen with atrial pacing, which are relieved by the administration of nitroglycerin, except in the presence of coronary collaterals.

Impact of echocardiography on the management of patients with mitral valve prolapse.

OBJECTIVE: To determine whether echocardiography affects the decisions to use beta blockers or to recommend bacterial endocarditis prophylaxis in patients suspected of having mitral valve prolapse (MVP). DESIGN: Retrospective review of echocardiograms and clinical records. SETTING: Military tertiary care hospital. PATIENTS: 127 patients with clinically suspected MVP (105) or incidentally discovered MVP (22). MAIN RESULTS: Beta blockers were used more often in patients with suspected MVP and positive echocardiograms (45%) than in patients with normal echocardiograms (13%, p less than 0.001). Bacterial endocarditis prophylaxis was recommended more often in patients with suspected MVP and positive echocardiograms (65%) than in patients with normal echocardiograms (11%, p less than 0.001). Presence or absence of a murmur did not influence the decision to recommend bacterial endocarditis prophylaxis. Patients in whom MVP was incidentally discovered were unlikely to receive either beta blockers or the recommendation for bacterial endocarditis prophylaxis. CONCLUSIONS: The results of echocardiography affect the decisions to use beta blockers or to recommend bacterial endocarditis prophylaxis in patients with suspected MVP.

Mitral valve prolapse: comparison of diagnosis by physical examination and echocardiography.

To determine how well physical examination findings suggestive of mitral valve prolapse (MVP) correlate with echocardiographic evidence of MVP, we retrospectively reviewed the charts of 104 patients referred to an Air Force Cardiology Clinic for echocardiography to rule out MVP. In each case, the referring physician's specialty and his findings on cardiac physical examination were recorded. All patients had M-mode echocardiography, and half of the patients had two-dimensional echocardiography. Sensitivities, specificities, and likelihood ratios for the physical examination were calculated using echocardiography as the comparison standard. The combination of a systolic click and a systolic murmur was the physical examination finding most predictive of echocardiographic MVP, with a positive likelihood ratio of 2.43. Other combinations of physical findings yielded likelihood ratios close to 1. No differences were found based on the specialty of the examining physician. We conclude that when practicing physicians find a systolic click and murmur, MVP is likely to be present on echocardiography, though one third of the patients will have normal echocardiograms. Other combinations of physical findings are of little help in predicting echocardiographic MVP.

Employment chances of recently visually impaired, young or middle-aged adults in the Swedish labour market.

The study (n = 261) focused on employment chances of Swedish visually impaired persons aged 25-45 years at the onset of their visual impairment, and who were employed at that time. It concerned persons who became visually impaired after 1974, the year when the Act on Security of Employment was passed. Although the Act has had an impact on employment opportunities, it is, however, no guarantee per se. Factors in the work environment accounted for improved chances of keeping the pre-disability employment. Individual factors were decisive in a person's chances of obtaining new employment. The least competitive groups of visually impaired were, in spite of the law, 'eliminated' from the labour market.

Hugging balloon coronary angioplasty of a large left circumflex coronary artery.

A case is described in which hugging balloons were used to perform coronary angioplasty through a single catheter. A discussion concerning selection of balloon combinations and technique is included.