Margaret M. Parker

Septic Shock in Humans: Advances in the Understanding of Pathogenesis, Cardiovascular Dysfunction, and Therapy

Septic shock is the commonest cause of death in intensive care units. Although sepsis usually produces a low systemic vascular resistance and elevated cardiac output, strong evidence (decreased ejection fraction and reduced response to fluid administration) suggests that the ventricular myocardium is depressed and the ventricle dilated. In survivors, these abnormalities are reversible. Failure to develop ventricular dilatation in nonsurvivors suggests that dilatation is a compensatory mechanism needed to maintain adequate cardiac output. With a canine model of septic shock that is very similar to human sepsis, myocardial depression was confirmed using load-independent measures of ventricular performance. Endotoxin administration to humans simulates the qualitative, cardiovascular abnormalities of sepsis. The pathogenesis of septic shock is extraordinarily complex. Diverse microorganisms can generate toxins, stimulating release of potent mediators that act on vasculature and myocardium. A circulating myocardial depressant substance has been closely associated with the myocardial depression of human septic shock. Therapy has emphasized early use of antibiotics, critical care monitoring, aggressive volume resuscitation, and, if shock continues, use of inotropic agents and vasopressors. Pharmacologic or immunologic antagonism of endotoxin or other mediators may prove to enhance survival in this highly lethal syndrome.

The cardiovascular response of normal humans to the administration of endotoxin.

Marked abnormalities in cardiovascular function accompany septic shock, and bacterial endotoxin is believed to be one of the principal mediators of these abnormalities. To evaluate the cardiovascular effects of endotoxemia in humans, we measured hemodynamic variables in nine normal subjects given an intravenous bolus dose of endotoxin (Escherichia coli, 4 ng per kilogram of body weight) and in six normal subjects given a bolus dose of saline, before and three hours after administration. All the subjects then underwent volume loading with normal saline (mean, 2217 ml) during the fourth and fifth hours after administration of the bolus, and the measurements were repeated. Three hours after the administration of endotoxin and before volume loading, the cardiac index had increased by 53 percent and the heart rate by 36 percent (both changes were significant; P less than or equal to 0.008), and the systemic vascular-resistance index had decreased by 46 percent (P = 0.004). After volume loading (five hours after the administration of endotoxin), the left ventricular ejection fraction decreased by 1 percent of the base-line value in the subjects given endotoxin, but increased by 14 percent in the controls (P = 0.008). The left ventricular end-diastolic and end-systolic volume indexes increased by 18 percent (P = 0.07) and 24 percent (P = 0.042), respectively. Left ventricular performance, as measured by the ratio of the peak systolic pressure to the end-systolic volume index, was depressed (a decrease of 0.90 in the subjects given endotoxin vs. an increase of 0.76 in the controls; P = 0.024). We conclude that the administration of endotoxin to normal subjects causes a depression of left ventricular function that is independent of changes in left ventricular volume or vascular resistance. The changes in function are similar to those observed in septic shock and suggest that endotoxin is a major mediator of the cardiovascular dysfunction in this condition.

Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC)

Advanced age-related macular degeneration (AMD) is the leading cause of late onset blindness. We present results of a genome-wide association study of 979 advanced AMD cases and 1,709 controls using the Affymetrix 6.0 platform with replication in seven additional cohorts (totaling 5,789 unrelated cases and 4,234 unrelated controls). We also present a comprehensive analysis of copy-number variations and polymorphisms for AMD. Our discovery data implicated the association between AMD and a variant in the hepatic lipase gene (LIPC) in the high-density lipoprotein cholesterol (HDL) pathway (discovery P = 4.53e-05 for rs493258). Our LIPC association was strongest for a functional promoter variant, rs10468017, (P = 1.34e-08), that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD. The association we found with LIPC was corroborated by the Michigan/Penn/Mayo genome-wide association study; the locus near the tissue inhibitor of metalloproteinase 3 was corroborated by our replication cohort for rs9621532 with P = 3.71e-09. We observed weaker associations with other HDL loci (ABCA1, P = 9.73e-04; cholesterylester transfer protein, P = 1.41e-03; FADS1-3, P = 2.69e-02). Based on a lack of consistent association between HDL increasing alleles and AMD risk, the LIPC association may not be the result of an effect on HDL levels, but it could represent a pleiotropic effect of the same functional component. Results implicate different biologic pathways than previously reported and provide new avenues for prevention and treatment of AMD.

Clinical InvestigationsDepressed Left Ventricular Performance: Response to Volume Infusion in Patients with Sepsis and Septic Shock

Volume infusion, to increase preload and to enhance ventricular performance, is accepted as initial management of septic shock. Recent evidence has demonstrated depressed myocardial function in human septic shock. We analyzed left ventricular performance during volume infusion using serial data from simultaneously obtained pulmonary artery catheter hemodynamic measurements and radionuclide cineangiography. Critically ill control subjects (n = 14), patients with sepsis but without shock (n = 21), and patients with septic shock (n = 21) had prevolume infusion hemodynamic measurements determined and received statistically similar volumes of fluid resulting in similar increases in pulmonary capillary wedge pressure. There was a strong trend (p = 0.004) toward less of a change in left ventricular stroke work index (LVSWI) after volume infusion in patients with sepsis and septic shock compared with control subjects. The LVSWI response after volume infusion was significantly less in patients with septic shock when compared with critically ill control subjects (p less than 0.05). These data demonstrate significantly altered ventricular performance, as measured by LVSWI, in response to volume infusion in patients with septic shock.

High-dose ifosfamide is associated with severe, reversible cardiac dysfunction

Ifosfamide is an oxazaphosphorine nitrogen mustard compound that is structurally similar to cyclophosphamide. The drug undergoes cytochrome P-450 microsomal activation and is excreted by the kidneys [1-4]. In clinical trials over the last three decades, ifosfamide has shown efficacy in the treatment of lymphomas; sarcomas; and testicular, breast, and lung carcinomas [5-9]. Renal, bone marrow, central nervous system, gastrointestinal, and bladder toxicities associated with ifosfamide have been well characterized in adults and in children [3, 10-16]. In one study, cardiac arrhythmias were observed in patients receiving ifosfamide therapy [17]. In another study, two patients who had been treated with doxorubicin and who subsequently received high-dose ifosfamide therapy developed congestive heart failure [6]. Except in these two studies, significant cardiovascular toxicities have not previously been associated with ifosfamide. We describe the cardiovascular complications in a group of patients who received ifosfamide as a component of combination chemotherapy with autologous bone marrow transplantation for treatment of lymphoma or carcinoma. Table. SI Unit and Abbreviations Methods Patients and Chemotherapy Regimens The medical records of all patients who received combination chemotherapy and autologous bone marrow transplantation at the National Institutes of Health between March 1988 and April 1991 were retrospectively reviewed. One group of patients was enrolled in a phase I study of high-dose combination chemotherapy and autologous bone marrow transplantation for treatment of metastatic carcinoma and lymphoma. These patients received ifosfamide (total dose, 10 to 18 g/m2 body surface area) with carboplatin (total dose, 900 to 1800 mg/m2) and etoposide (total dose, 600 to 1800 mg/m2). This combination is referred to as the ICE (ifosfamide, carboplatin, and etoposide) regimen [18]. The other group of patients participated in a phase I study of salvage chemotherapy followed by intensification chemotherapy and autologous bone marrow transplantation for refractory Hodgkin disease. These patients received ifosfamide (total dose, 10 to 16 g/m2) together with vinblastine (total dose, 16 to 25 mg/m2) and lomustine (also known as CCNU [N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea], total dose, 240 to 320 mg/m2). This combination is referred to as the VIC (vinblastine, ifosfamide, and CCNU) regimen [19]. In both regimens, ifosfamide was given with mesna doses ranging from 16 to 28.8 g/m2. In both phase I studies, the total amount of ifosfamide was divided into equal doses administered intravenously for 2 hours each day for 4 consecutive days. Mesna, admixed with ifosfamide, was administered as a loading dose over a 2-hour period, and this was followed immediately by a 3-hour continuous infusion. After completion of the infusion, mesna was administered intravenously every 3 hours for 15 minutes for a total of six doses. Carboplatin was administered as a 72-hour continuous infusion; etoposide was given intravenously every 12 hours in six divided doses; and vinblastine was given as an intravenous loading dose followed by a continuous infusion for 96 hours. Lomustine was given orally as two consecutive daily doses. Bone marrow that had been previously harvested was reinfused on day 7 in the ICE regimen and on day 9 in the VIC regimen. All patients were hydrated intravenously from 24 hours before the first dose of ifosfamide until at least 7 days after completion of ifosfamide therapy. Patients on the ICE regimen received 5% dextrose and 0.9% sodium chloride at 300 mL/h, and those on the VIC regimen received 5% dextrose and 0.45% sodium chloride solution at 3 L/m2 per 24 hours. Furosemide was given as needed to maintain urine output of at least 150 mL/h in all patients. Retrospective Chart Review The following clinical and laboratory data were reviewed: details of daily physical examinations; electrocardiograms, echocardiograms, radionuclide cineangiograms, and chest radiographs; serial measurements of serum creatinine, minerals, electrolytes, and arterial pH and blood gases; and results of all cultures obtained during hospitalization. Hemodynamic Monitoring and Calculations Patients admitted to the intensive care unit with congestive heart failure underwent hemodynamic monitoring with pulmonary artery catheters (Sorenson, 7.5 F, Abbott Critical Care Systems, North Chicago, Illinois) and intra-arterial catheters (20 G, 4.5 cm, Arrow International, Reading, Pennsylvania). Hemodynamic variables assessed serially in the intensive care unit included heart rate; mean arterial pressure; central venous pressure; pulmonary artery systolic, diastolic, and mean pressures; and pulmonary artery occlusion pressure. Cardiac output was determined by thermodilution technique. The cardiac index was determined by dividing cardiac output by body surface area. At the time of admission to the intensive care unit, left ventricular ejection fraction was obtained by bedside radionuclide cineangiography (Picker portable camera, model Dynamo, Northford, Connecticut) using standard techniques [20]. Echocardiograms were obtained using a 2.5-MHz transducer connected to a Hewlett-Packard ultrasound system. Left ventricular fractional shortening was calculated by dividing the difference between the end-diastolic and end-systolic dimensions by the end-diastolic dimension. Histopathologic Evaluation For cases in which autopsy material was available, gross pathologic and histopathologic findings in the heart and cardiovascular system were reviewed and noted. Statistical Methods Data on the following variables were collected for each of the eight patients admitted to the intensive care unit: weight, heart rate, mean arterial pressure, pulmonary artery mean pressure, pulmonary artery occlusion pressure, cardiac index, left ventricular ejection fraction, and fractional shortening. For each variable, the mean value at the time of admission to the intensive care unit was compared with that at follow-up (day 4) by a paired sample t-test. Unpaired Student t-tests were used to compare means of previous cumulative doses of doxorubicin. The Yates mean score test [21] was done to evaluate the association between ifosfamide dose and the occurrence of congestive heart failure. A Fisher exact test was used to assess the correlation of congestive heart failure with doubling of serum creatinine level, presence or absence of previous doxorubicin therapy, and presence or absence of underlying lymphoma. Results During the period reviewed, 52 patients received 53 courses of chemotherapy in association with autologous bone marrow transplantation. This group included 22 women and 30 men with a mean age of 36 years (range, 15 to 65 years). Their primary diagnoses were Hodgkin disease (n = 24), diffuse large cell lymphoma (n = 10), other intermediate- or high-grade lymphomas (n = 7), breast carcinoma (n = 7), and testicular carcinoma (n = 4). The ifosfamide dose was 10 g/m2 in 6 patients, 12.5 g/m2 in 12 patients, 15.6 g/m2 in 20 patients, 16 g/m2 in 12 patients, and 18 g/m2 in 3 patients. Nineteen patients received the VIC regimen, and 34 received the ICE regimen. One patient received both regimens, initially VIC with an ifosfamide dose of 15.6 g/m2, and, after relapse 13 months later, ICE with an ifosfamide dose of 16 g/m2. Before ifosfamide therapy, 45 patients had received doxorubicin in cumulative doses (mean SE) of 384 23 mg/m2 (range, 45 to 650 mg/m2). Twenty patients had received radiation therapy to the chest or mediastinum at times ranging from 1 week to 10 years before ifosfamide therapy. None of the 52 patients had a previous history of congestive heart failure, angina pectoris, or cardiac arrhythmias. At the time of admission, all patients had normal cardiovascular findings at physical examination. All patients had serum creatinine levels below 130 mol/L (1.5 mg/dL) and creatinine clearances above 50 mL/min before initiation of therapy. Nine of the 52 patients developed significant cardiovascular toxicity yielding an overall incidence of 17% (95% CI, 8% to 30%). Of 34 patients given the ICE regimen (23 with lymphoma and 11 with carcinoma), 6 patients with lymphoma (18%) developed congestive heart failure. Of 19 patients with lymphoma on the VIC regimen, 3 (16%) developed congestive heart failure. A significant dose-response trend was observed in the incidence of congestive heart failure as the ifosfamide dose was increased (P = 0.02): Congestive heart failure developed in 0 of 6 patients receiving 10 g/m2, 1 of 12 patients (8%) receiving 12.5 g/m2, 2 of 20 patients (10%) receiving 15.6 g/m2, 4 of 12 patients (33%) receiving 16 g/m2, and 2 of 3 patients (67%) receiving 18 g/m2. Eight of the nine patients developed severe congestive heart failure and required admission to the intensive care unit. Demographic data for these eight patients are shown in Table 1. Three patients had received mediastinal radiation therapy. All eight patients had received previous doxorubicin therapy (mean cumulative dose, 340 mg/m2; range, 190 to 550 mg/m2) at a median interval of 1 month (range, 0.5 to 36 months) before the administration of ifosfamide. Another 26 patients who received ifosfamide doses of 15.6 to 18 g/m2 had also received previous therapy with doxorubicin at a similar mean cumulative dose (326 mg/m2 [range, 45 to 650 mg/m2]; P > 0.2) and at a similar median interval of 1 month (range, 0.33 to 4.6 months) before ifosfamide therapy; however, these patients did not develop serious cardiac complications. Table 1. Demographic and Clinical Characteristics of Eight Patients Admitted to the Intensive Care Unit with Congestive Heart Failure after Ifosfamide Therapy* The onset of symptoms of heart failure occurred at a mean of 12 days (range, 6 to 23 days) after the initiation of ifosfamide therapy. By the time of admission to the intensive care unit, all eight patients had dyspnea, t

Confirming genes influencing risk to cleft lip with/without cleft palate in a case–parent trio study

A collection of 1,108 case–parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525–529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as ‘second tier’ hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene–environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene–gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Identification of Functional Variants for Cleft Lip with or without Cleft Palate in or near PAX7, FGFR2, and NOG by Targeted Sequencing of GWAS Loci

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

Peak systolic pressure/end-systolic volume ratio, a load-independent measure of ventricular function, is reversibly decreased in human septic shock.

Objective: To evaluate left ventricular performance in patients with septic shock, using a load‐independent measure of left ventricular systolic function, the peak systolic pressure/ end‐systolic volume index ratio. Setting: National Institutes of Health research facility. Design: Prospective study with retrospective analysis of data. Patients: Twenty‐seven patients with septic shock, 13 critically ill, nonseptic control patients, and nine normal volunteers. Measurements: Hemodynamic measurements from indwelling arterial and pulmonary artery catheters and radionuclide‐determined ejection fraction measurements. These data were used to calculate the peak systolic pressure/end‐systolic volume index ratio in each patient group. The survivors and nonsurvivors of septic shock were evaluated on admission to the intensive care unit and at recovery in the survivors or within 24 hrs of death in the nonsurvivors. The ratio in each group was compared with the ratio in the critically ill, nonseptic patients and the normal volunteers. Main Results: Both survivors and non‐survivors of septic shock had a decreased peak systolic pressure/end‐systolic volume index ratio (1.2 ± 0.1 and 1.7 ± 0.2 mm Hg/mL/m2, respectively) compared with critically ill, nonseptic patients (3.5 ± 0.7 mm Hg/mL/m2; both p < .05) and normal volunteers (4.3 ± 0.4 mm Hg/mL/m2; both p < .05). The differences between the groups were highly significant ( p < .001) by analysis of variance. The survivors had a lower initial peak systolic pressure/end‐systolic volume index ratio than the nonsurvivors ( p < .05). In the survivors, the peak systolic pressure/end‐systolic volume index ratio increased significantly ( p < .05) with recovery to 2.4 ± 0.3 mm Hg/mL/m2. Serial determinations of peak systolic pressure/end‐systolic volume index ratio in the nonsurvivors did not show any significant change. Conclusions: This study confirms that survivors and nonsurvivors of septic shock have significant depression of myocardial performance as measured by a load‐independent technique. Survivors have greater depression of myocardial performance than nonsurvivors, and, with recovery, the ventricular performance in survivors increases toward normal values. (Crit Care Med 1994; 22:1955–1959)

Whole Exome Sequencing of Distant Relatives in Multiplex Families Implicates Rare Variants in Candidate Genes for Oral Clefts

A dozen genes/regions have been confirmed as genetic risk factors for oral clefts in human association and linkage studies, and animal models argue even more genes may be involved. Genomic sequencing studies should identify specific causal variants and may reveal additional genes as influencing risk to oral clefts, which have a complex and heterogeneous etiology. We conducted a whole exome sequencing (WES) study to search for potentially causal variants using affected relatives drawn from multiplex cleft families. Two or three affected second, third, and higher degree relatives from 55 multiplex families were sequenced. We examined rare single nucleotide variants (SNVs) shared by affected relatives in 348 recognized candidate genes. Exact probabilities that affected relatives would share these rare variants were calculated, given pedigree structures, and corrected for the number of variants tested. Five novel and potentially damaging SNVs shared by affected distant relatives were found and confirmed by Sanger sequencing. One damaging SNV in CDH1, shared by three affected second cousins from a single family, attained statistical significance (P = 0.02 after correcting for multiple tests). Family-based designs such as the one used in this WES study offer important advantages for identifying genes likely to be causing complex and heterogeneous disorders.

A genome-wide association study identifies risk loci for spirometric measures among smokers of European and African ancestry

BackgroundPulmonary function decline is a major contributor to morbidity and mortality among smokers. Post bronchodilator FEV1 and FEV1/FVC ratio are considered the standard assessment of airflow obstruction. We performed a genome-wide association study (GWAS) in 9919 current and former smokers in the COPDGene study (6659 non-Hispanic Whites [NHW] and 3260 African Americans [AA]) to identify associations with spirometric measures (post-bronchodilator FEV1 and FEV1/FVC). We also conducted meta-analysis of FEV1 and FEV1/FVC GWAS in the COPDGene, ECLIPSE, and GenKOLS cohorts (total n = 13,532).ResultsAmong NHW in the COPDGene cohort, both measures of pulmonary function were significantly associated with SNPs at the 15q25 locus [containing CHRNA3/5, AGPHD1, IREB2, CHRNB4] (lowest p-value = 2.17 × 10−11), and FEV1/FVC was associated with a genomic region on chromosome 4 [upstream of HHIP] (lowest p-value = 5.94 × 10−10); both regions have been previously associated with COPD. For the meta-analysis, in addition to confirming associations to the regions near CHRNA3/5 and HHIP, genome-wide significant associations were identified for FEV1 on chromosome 1 [TGFB2] (p-value = 8.99 × 10−9), 9 [DBH] (p-value = 9.69 × 10−9) and 19 [CYP2A6/7] (p-value = 3.49 × 10−8) and for FEV1/FVC on chromosome 1 [TGFB2] (p-value = 8.99 × 10−9), 4 [FAM13A] (p-value = 3.88 × 10−12), 11 [MMP3/12] (p-value = 3.29 × 10−10) and 14 [RIN3] (p-value = 5.64 × 10−9).ConclusionsIn a large genome-wide association study of lung function in smokers, we found genome-wide significant associations at several previously described loci with lung function or COPD. We additionally identified a novel genome-wide significant locus with FEV1 on chromosome 9 [DBH] in a meta-analysis of three study populations.