Mechanical Forces Govern Interactions of Host Cells with Intracellular Bacterial Pathogens.

To combat infectious diseases, it is important to understand how host cells interact with bacterial pathogens. Signals conveyed from pathogen to host, and vice versa, may be either chemical or mechanical. While the molecular and biochemical basis of host-pathogen interactions has been extensively explored, relatively less is known about mechanical signals and responses in the context of those interactions. Nevertheless, a wide variety of bacterial pathogens appear to have developed mechanisms to alter the cellular biomechanics of their hosts in order to promote their survival and dissemination, and in turn many host responses to infection rely on mechanical alterations in host cells and tissues to limit the spread of infection. In this review, we present recent findings on how mechanical forces generated by host cells can promote or obstruct the dissemination of intracellular bacterial pathogens. In addition, we discuss how in vivo extracellular mechanical signals influence interactions between host cells and intracellular bacterial pathogens. Examples of such signals include shear stresses caused by fluid flow over the surface of cells and variable stiffness of the extracellular matrix on which cells are anchored. We highlight bioengineering-inspired tools and techniques that can be used to measure host cell mechanics during infection. These allow for the interrogation of how mechanical signals can modulate infection alongside biochemical signals. We hope that this review will inspire the microbiology community to embrace those tools in future studies so that host cell biomechanics can be more readily explored in the context of infection studies.

Volume measurement and biophysical characterization of mounds in epithelial monolayers after intracellular bacterial infection.

Mechanical forces are important in (patho)physiological processes, including how host epithelial cells interact with intracellular bacterial pathogens. As these pathogens disseminate within host epithelial monolayers, large mounds of infected cells are formed due to the forceful action of surrounding uninfected cells, limiting bacterial spread across the basal cell monolayer. Here, we present a protocol for mound volume measurement and biophysical characterization of mound formation. Modifications to this protocol may be necessary for studying different host cell types or pathogenic organisms. For complete details on the use and execution of this protocol, please refer to Bastounis et al. (2021).

Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially infected epithelial cells.

Intracellular pathogens alter their host cells' mechanics to promote dissemination through tissues. Conversely, host cells may respond to the presence of pathogens by altering their mechanics to limit infection. Here, we monitored epithelial cell monolayers infected with intracellular bacterial pathogens, Listeria monocytogenes or Rickettsia parkeri, over days. Under conditions in which these pathogens trigger innate immune signaling through NF-κB and use actin-based motility to spread non-lytically intercellularly, we found that infected cell domains formed three-dimensional mounds. These mounds resulted from uninfected cells moving toward the infection site, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the infection focus, while extruded infected cells underwent cell death. Thus, the coordinated forceful action of uninfected cells actively eliminates large domains of infected cells, consistent with this collective cell response representing an innate immunity-driven process.

Combination of "Markers" and the Consequent Importance of Fetal Abdominal Circumference in the First Trimester for Predicting Complications Specific to Monochorionic Twin Pregnancies.

BACKGROUND: Multifetal pregnancies are considered high-risk pregnancies compared to singleton pregnancies. Monochorionic diamniotic (MCDA) twin pregnancies tend to have a more complicated intrauterine course than their dichorionic counterparts. The most common complications are twin-to-twin transfusion syndrome (TTTS), selective fetal growth restriction (sFGR), and unexplained intrauterine fetal demise (IUFD). Early recognition of pregnancies at risk of developing complications will be helpful for counselling the parents and preparing them for necessary interventions in case a complication arises. OBJECTIVES: The aim of our study was to assess the role of discordant crown-rump length (dCRL), discordant nuchal translucency (dNT), and discordant abdominal circumference (dAC) measurements in isolation and in combination at the 11-13+6 weeks' scan in predicting the development of TTTS, sFGR, and IUFD. METHODS: All MCDA twin pregnancies with structurally normal, both live fetuses at the 11-13+6 weeks' scan were followed up by FMF (Fetal Medicine Foundation)-certified operators for development of TTTS, sFGR, and single/double IUFD until delivery. Discordance of CRL, NT, and AC of more than or equal to 10% was considered a "marker," and its presence was correlated with the occurrence of specific antenatal complications, i.e., TTTS, sFGR, and single or double IUFD. RESULTS: Combination of two markers predicted MCDA-related complications in the majority of cases (70.8% positive predictive value). Combination of dNT and dAC was the strongest predictor (80% positive predictive value). CONCLUSIONS: In our population of MC twin pregnancies, dCRL, dNT, and dAC at the 11+0-13+6 weeks' scan, in isolation, had a low predictive value for antenatal complications, i.e., TTTS, sFGR, and single/double IUFD. However, the presence of at least two markers in the first trimester could predict complications in the majority of these pregnancies. The combination of dNT and dAC appears to be the best.

Prediction of Fetal Anemia in Subsequent Transfusions: Is There a Need to Change the Threshold of the Peak Systolic Velocity of the Middle Cerebral Artery?

INTRODUCTION: Peak systolic velocity (PSV) of the middle cerebral artery (MCA) shows 100% sensitivity for predicting fetal anemia before the first intrauterine transfusion (IUT). However, its ability to predict subsequent transfusions has remained mostly controversial. OBJECTIVES: To assess if there is a need to change the threshold of MCA-PSV from 1.5 to 1.69 multiples of the median (MoM) to predict fetal anemia and the need for subsequent IUT. METHODS: This is a retrospective audit, wherein case records of mothers who underwent IUT at the Bangalore Fetal Medicine Centre between April 2008 and May 2017 were reviewed; 86 cases were included, and the data were analyzed using MS Excel. The MCA-PSV and pretransfusion Hb were converted into MoM. 40 fetuses that had more than 1 IUT were included in the analysis. -Results: 31/40 fetuses that had >1 IUT had an MCA-PSV >1.5 MoM, of which 29 were anemic according to the post-IUT Hb MoM. 20/29 (69%) had an MCA-PSV >1.69, whereas 9/29 (31%) had an MCA-PSV between 1.5 and 1.69 MoM. Our study shows that changing the MCA-PSV threshold from 1.5 to 1.69 MoM will reduce the detection of fetal anemia and hence the need for repeat IUT by 31%. CONCLUSIONS: Increasing the fetal MCA-PSV threshold from 1.5 to 1.69 will miss out one-third of the fetuses that will require a 2nd, 3rd, or 4th IUT. This is more relevant in geographical areas where the parents must travel long distances for IUTs, which are performed in tertiary fetal care centers.

Outcome of Pregnancies Diagnosed with TRAP Sequence Prenatally: A Single-Centre Experience.

INTRODUCTION: Twin reversed arterial perfusion (TRAP) sequence is a phenomenon seen in 1-3% of monochorionic twin pregnancies, where the acardiac fetus is found to have multiple anomalies. The normal pump twin maintains its own perfusion in addition to that of its acardiac co-twin. As a result, if the acardiac twin increases in size, the burden on the pump twin is increased, leading to cardiac failure, hydrops fetalis, polyhydramnios, premature delivery, and perinatal death. The outcome of pregnancy is largely dependent on the growth of the acardiac twin. In view of high perinatal loss, surgical techniques to interrupt the blood flow to the acardiac twin have been evaluated. METHODS: We evaluated interstitial laser therapy, which is the main mode of therapy in our unit, of TRAP pregnancies referred over a 5-year period. Interstitial laser was offered if the blood flow in the acardiac twin was found to be persistent at 2 consecutive examinations or if there were cardiac or hydropic changes in the pump twin at the first examination. RESULTS: A total of 18 cases of TRAP were referred during this period and all were counselled regarding fetal therapy if the situation were to deteriorate; 5 couples (27.7%) opted for termination of pregnancy; of the remaining 13, 7 (53.8%) agreed to perform intervention following confirmation of a normal karyotype. Six (85.7%) and 1 (14.3%) lasers were performed in the second and third trimesters, respectively; all 7 had a normal outcome of the pump twin. There were 6/13 (46.2%) in the expectant group who continued the pregnancy with no intervention, with 2 term live births (33.3%). CONCLUSIONS: Our study confirms that there is a high risk of spontaneous loss in untreated pregnancies with TRAP, primarily due to polyhydramnios and fetal hydrops. In the pregnancies that underwent interstitial laser, there was a more favourable outcome. Interstitial laser is minimally invasive, safe, and feasible in experienced hands.

Performance of Common Down Syndrome Screening Methods Used in India with Construction of an Indian Normogram for Nuchal Translucency/Crown-Rump Length Measurements in 14,337 Subjects.

BACKGROUND/PURPOSE OF STUDY: Sonologists often lack access to the Fetal Medicine Foundation Down syndrome risk calculation software or cannot offer the combined test for aneuploidy screening because of resource constraints. Instead, sonologists use nuchal translucency (NT) measurements that rely on fixed NT cut-offs for labelling foetuses with high risks for Down syndrome. In the present study, we aimed to plot normative data for NT and the crown-rump length (CRL) in Indian foetuses and to assess the value of using the 95th/99th centiles of NT for CRL cut-offs instead of fixed NT cut-offs to calculate the risk for aneuploidies during the first trimester. METHODS: We conducted a retrospective observational study measuring the NT/CRL in 14,337 Indian foetuses between 11 and 13 + 6 weeks of gestation. We used regression analysis and calculated the 95th/99th centiles of NT distribution. We compared performances of NT > 95th/99th centiles and fixed NT cut-offs of 2.5- and 3.5-mm  as screening. RESULTS: The NT measurements increased with increasing CRL values. NT > 95th centile for a particular CRL for detecting all aneuploidies had the maximum sensitivity of 73.9% for a false positive rate (FPR) of 4.3%. Similar values for fixed cut-off > 2.5 mm were 63.0% FPR 3.7% (T21 68.2 FPR 3.8%) and for NT > 3.5 mm 36.9% FPR 0.43% (T21 36.5% FPR 0.5%). CONCLUSION: A fixed NT cut-off point is not appropriate for the first trimester screening. The best sensitivity for assessing aneuploidies is achieved using the 95th centile, but the 99th centile achieves higher specificity for gestational age.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required.

Anaesthesia for foetoscopic Laser ablation- a retrospective study.

BACKGROUND AND AIMS: Twin pregnancy with monochorionic placenta may be associated with arteriovenous vascular anastomosis of the placental vessels resulting in twin-to-twin transfusion syndrome (TTTS) and twin reversed arterial perfusion syndrome (TRAP). Foetoscopic LASER ablation (FLA) is the treatment of choice in reducing foetal mortality related to this. METHODS: A retrospective review of medical records of 41 FLA procedures for TTTS and TRAP were analysed for anaesthetic management. Thirty-four patients received subarachnoid block, three combined spinal-epidural block, three general anaesthesia and one local anaesthesia with sedation. Nitroglycerine 5 mg patch was used for tocolysis 1 h before the procedure and continued for 24-48 h postoperatively. RESULTS: Bupivacaine was used in 34 patients, and ropivacaine in three patients. Mean dose of bupivacaine 0.5% was 2.43 ± 0.32 ml and ropivacaine 0.75% was 2.85 ± 0.19 ml. The mean duration of surgery was 117.07 ± 28 min. Mild hypotension occurred in all patients under spinal anaesthesia and was treated with vasopressors. The foetal outcome among all 41 patients were 13 delivered live twins, 15 had a single live baby with intrauterine death of other twin baby. In 12 patients, both babies were intrauterine death. One patient was lost for follow-up. CONCLUSION: Foetoscopic procedures can be done under central neuraxial block, however occasionally general anaesthesia may be required.

Nasal bone length: the long and short of it. Evaluation of the reference values for the fetal nasal bone length at 16 to 25 weeks of gestational age in an Indian population.

OBJECTIVE: To establish a reference interval range for the fetal nasal bone length (NBL) in an Indian population. METHODS: A retrospective study of antenatal ultrasound records of fetuses at 16 to 25 weeks in singleton pregnancies. NBL, biparietal diameter (BPD) and femur length were noted in 6436 observations of singleton, euploid, nonmalformed pregnancies. RESULTS: The 50th centile for NBLs increased from 3.4 to 6.9 mm from 16th to 25th week of gestational age. The relation between nasal bone and gestational age, BPD and femur length were found and noted. The NBLs were found to be shorter when compared with other ethnicities. CONCLUSIONS: A reference range for fetal NBLs at 16 to 25 weeks in an Indian population and its relation with fetal biometry was established.

Mid-second Trimester Measurement of Nasal Bone Length in the Indian Population.

OBJECTIVE: The purpose of this study was to establish an Indian reference for normal fetal nasal bone length at 16-26 weeks of gestation. METHODS: The fetal nasal bone was measured by ultrasound in 2,962 pregnant women at 16-26 weeks of gestation from 2004 to 2009 by a single operator, who performed three measurements for each woman when the fetus was in the midsagittal plane and the nasal bone was between a 45 and 135° angle to the ultrasound beam. All neonates were examined after delivery to confirm the absence of congenital abnormalities. RESULTS: The median nasal bone length increased with gestational age from 3.3 mm at 16 weeks to 6.65 mm at 26 weeks in a linear relationship. The fifth percentile nasal bone lengths were 2.37, 2.4, 2.8, 3.5, 3.6, 3.9, 4.3, 4.6, 4.68, 4.54, and 4.91 mm at 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 weeks, respectively. CONCLUSIONS: We have established the nasal bone length in South Indian fetuses at 16-26 weeks of gestation and there is progressive increase in the fifth percentile of nasal bone length with advancing gestational age. Hence, gestational age should be considered while defining hypoplasia of the nasal bone.