Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive RNA-seq atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in-vivo is prevalent in diverse tissues. Unexpectedly, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.This research was supported by the EU-FP7-PEOPLE career integration grant, the Israeli Science Foundation (1073/14; N.S.-G.), Infect-ERA (TANKACY; N.S.-G.), the European Research Council starting grant (StG-2014-638142; N.S.-G.), the British Medical Research Programme (grant G0701279; J.S.), a Wellcome Research Studentship Grant (B.K.), and the Cambridge NIHR BRC Cell Phenotyping Hub. N.S.-G. is incumbent of the Skirball career development chair in new scientist

Elevated CPK levels after hydrazine inhalation exposure in an F16 aircraft technician

Hydrazine is a hazardous material that is commonly used in the pharmaceutical industry, as well as in rocket and jet fuels, including the emergency power unit of F-16 model jets. We present four ground crew technicians who were exposed to hydrazine for less than one minute, due to a voltage fall in an F-16 jet. Physical examinations were normal and none of the technicians were symptomatic for toxicity. One of the technicians had abnormal blood chemistry levels for liver and muscle enzymes: serum glutamic-oxaloacetic transaminase(SGOT)-321U/L, serum glutamate-pyruvate transaminase (SGPT)-123U/L, and creatine phosphokinase (CPK) 3300U/L. The CPK level peaked during hospitalization to 20960U/L at 36 h after the exposure, and subsequently declined. Upon release from the hospital, 48 h after the exposure, the CPK level -was 9429U/L. In repeated tests one week and one year after exposure, liver function and CPK levels were normal. We conclude that evaluation of blood tests is important, in addition to a physical examination, in asymptomatic persons following exposure to even short term highly elevated levels of hydrazine. Keywords: Hydrazine, Toxicity, Creatine phosphokinas

A Nearly Lethal Screw: An Unusual Cause of Recurrent Bradycardia and Asystole Episodes after Fixation of the Cervical Spine

We present a case of a 51-year-old man who was injured in a bicycle accident. His main injury was an unstable fracture of the cervical and thoracic vertebral column. Several hours after his arrival to the hospital the patient underwent open reduction and internal fixation (ORIF) of the cervical and thoracic spine. The patient was hospitalized in our critical care unit for 99 days. During this time patient had several episodes of severe bradycardia and asystole; some were short with spontaneous return to sinus and some required pharmacological treatment and even Cardiopulmonary Resuscitation (CPR). Initially, these episodes were attributed to the high cervical spine injury, but, later on, CT scan suggested that a fixation screw abutted on the esophagus and activated the vagus nerve by direct pressure. After repositioning of the cervical fixation, the bradycardia and asystole episodes were no longer observed and the patient was released to a rehabilitation ward. This case is presented in order to alert practitioners to the possibility that, after operative fixation of cervical spine injuries, recurrent episodes of bradyarrhythmia can be caused by incorrect placement of the fixation screws and might be confused with the natural history of the high cervical cord injury

Potassium Level Variation Following Packed Cell Transfusion in Critically Ill Adult Patients—How Alert Should We Be?

One of the most clinically important effects following the administration of packed cell transfusion (PCT) is hyperkalemia, which can cause severe life-threatening cardiac arrhythmias. This retrospective population-based cohort study included adults hospitalized between January 2007 and December 2019 in a general intensive care unit for 24 h or more, with normal levels of serum potassium on admission. We assessed changes in serum potassium levels after administration of one unit of packed cells and sought to identify clinical parameters that may affect these changes. We applied adjusted linear mixed models to assess changes in serum potassium. The mean increase in serum potassium was 0.09 mEq/L (C.U 0.04–0.14, p-value < 0.001) among the 366 patients who were treated with a single PCT compared to those not treated with PCT. Increased serum potassium levels were also found in patients who required mechanical ventilation, and to a lesser degree in those treated with vasopressors. Hypertension, the occurrence of a cerebrovascular accident, and increased creatinine levels were all associated with reduced serum potassium levels. Due to the small rise in serum potassium levels following PCT, we do not suggest any particular follow-up measures for critically ill patients who receive PCT

Can the Duration of In-Hospital Ventilation in Patients with Sepsis Help Predict Long-Term Survival?

Mechanical ventilation is a cornerstone in the treatment of critical illness, especially sepsis. Prolonged mechanical ventilation, for a duration exceeding 21 days, is associated with higher rates of in-hospital and post-discharge mortality. Our aim was to assess the association between in-hospital ventilation duration and long-term life expectancy in patients ventilated in intensive care units specifically due to sepsis of any origin. We conducted a population-based retrospective cohort study of adults hospitalized in a general intensive care unit for 24 h or more during 2007–2017, who were diagnosed with sepsis or septic shock, treated with invasive mechanical ventilation for a maximum of 60 days and survived hospitalization. The primary exposure was the length of invasive mechanical ventilation. In an adjusted multivariable regression model, survival rates at 1, 2, 3 and 4 years post-hospitalization did not differ significantly between patients who were ventilated for 3–8 days (n = 169), 9–21 days (n = 160) or 22–60 days (n = 170), and those who were ventilated for 1–2 days (n = 192). We concluded that the duration of in-hospital ventilation in patients with sepsis cannot serve as a predictor for long-term survival. Thus, the duration of ventilation in itself should not guide the level of care in ventilated patients with sepsis

Estimation of Potassium Changes Following Potassium Supplements in Hypokalemic Critically Ill Adult Patients–A Patient Personalized Practical Treatment Formula

Hypokalemia is common among critically ill patients. Parenteral correction of hyperkalemia depends on dosages and patient characteristics. Our aims were to assess changes in potassium levels following parenteral administration, and to derive a formula for predicting rises in serum potassium based on patient characteristics. We conducted a population-based retrospective cohort study of adults hospitalized in a general intensive care unit for 24 h or more between December 2006 and December 2017, with hypokalemia. The primary exposures were absolute cumulative intravenous doses of 20, 40, 60 or 80 mEq potassium supplement. Adjusted linear mixed models were used to estimate changes in serum potassium. Of 683 patients, 422 had mild and 261 moderate hypokalemia (serum potassium 3.0–3.5 mEq/L and 2.5–2.99 mEq, respectively). Following doses of 20–80 mEq potassium, serum potassium levels rose by a mean 0.27 (±0.4) mEq/L and 0.45 (±0.54) mEq/L in patients with mild and moderate hypokalemia, respectively. Changes were associated with creatinine level, and the use of mechanical ventilation and vasopressors. Among critically ill patients with mild to moderate hypokalemia, increases in serum potassium after intravenous potassium supplement are influenced by several clinical parameters. We generated a formula to predict the expected rise in serum potassium based on clinical parameters

Challenging the Interpretation of White Blood Cell Counts in Patients with Sepsis Following Packed Cell Transfusion

Critically ill patients with sepsis often require packed cell transfusions (PCT). However, PCT may affect white blood cell (WBC) counts. We conducted a population-based retrospective cohort study to trace changes in WBC count following PCT in critically ill patients with sepsis. We included 962 patients who received one unit of PCT while hospitalized in a general intensive care unit, and 994 matched patients who did not receive PCT. We calculated the mean values of WBC count for the 24 h before and 24 h after PCT. Multivariable analyses using a mixed linear regression model were performed. The mean WBC count decreased in both groups, but more in the non-PCT group (from 13.9 × 109/L to 12.2 × 109/L versus 13.9 × 109/L to 12.8 × 109/L). A linear regression model showed a mean decrease of 0.45 × 109/L in WBC count over the 24 h following the start of PCT. Every 1.0 × 109/L increase in the WBC count prior to PCT administration showed a corresponding decrease of 0.19 × 109/L in the final WBC count. In conclusion, among critically ill patients with sepsis, PCT causes only mild and clinically non-prominent changes in WBC count

Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease

International audienceAmeloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta