Objective Assessment of Physiologic Alterations Associated With Hemodynamically Significant Patent Ductus Arteriosus in Extremely Premature Neonates.

Delay in closure of ductus arteriosus in postnatal life may lead to serious consequences and complications in an extremely premature neonate secondary to hemodynamic alterations in regional blood flow pattern in various organs. Despite the widespread recognition amongst neonatologists to identify a hemodynamically significant patent ductus arteriosus (hsPDA) early in the postnatal course, there is lack of consensus in its definition and thus the threshold to initiate treatment. Echocardiographic assessment of PDA shunt size and volume combined with neonatologists' impression of clinical significance is most frequently used to determine the need for treatment of PDA. Common clinical signs of hsPDA utilized as surrogate for decreased tissue perfusion may lag behind early echocardiographic signs. Although echocardiogram allows direct assessment of PDA shunt and hemodynamic alterations in the heart, it is limited by dependence on pediatric cardiologist availability, interobserver variation and isolated time point assessment. Electrical cardiometry (EC) is a non-invasive continuous real time measurement of cardiac output by applying changes in thoracic electrical impedance. EC has been validated in preterm newborns by concomitant transthoracic echocardiogram assessments and may be beneficial in studying changes in cardiac output in premature newborns with hsPDA. Alterations in perfusion index derived from continuous pulse oximetry monitoring has been used to study changes in cardiac performance and tissue perfusion in infants with PDA. Near infrared spectroscopy (NIRS) has been used to objectively and continuously assess variations in renal, mesenteric, and cerebral oxygen saturation and thus perfusion changes due to diastolic vascular steal from hsPDA in preterm neonates. Doppler ultrasound studies measuring resistive indices in cerebral circulation indicate disturbance in cerebral perfusion secondary to ductal steal. With recent trends of change in practice toward less intervention in care of preterm newborn, treatment strategy needs to be targeted for select preterm population most vulnerable to adverse hemodynamic effects of PDA. Integration of these novel ways of hemodynamic and tissue perfusion assessment in routine clinical care may help mitigate the challenges in defining and targeting treatment of hsPDA thereby improving outcomes in extremely premature neonates.

Ontogeny of inter-alpha inhibitor protein (IAIP) expression in human brain.

Inter-alpha inhibitor proteins (IAIPs) are naturally occurring immunomodulatory molecules found in most tissues. We have reported ontogenic changes in the expression of IAIPs in brain during development in sheep and abundant expression of IAIPs in fetal and neonatal rodent brain in a variety of cellular types and brain regions. Although a few studies identified bikunin, light chain of IAIPs, in adult human brain, the presence of the complete endogenous IAIP protein complex has not been reported in human brain. In this study, we examined the immunohistochemical expression of endogenous IAIPs in human cerebral cortex from early in development through the neonatal period and in adults using well-preserved postmortem brains. We examined total, nuclear, and cytoplasmic staining of endogenous IAIPs and their expression in neurofilament light polypeptide-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes. IAIPs were ubiquitously detected for the first time in cerebral cortical cells at 24-26, 27-28, 29-36, and 37-40 weeks of gestation and in adults. Quantitative analyses revealed that IAIPs were predominately localized in the nucleus in all age groups, but cytoplasmic IAIP expression was more abundant in adult than in the younger ages. Immunoreactivity of IAIPs was expressed in neurons and astrocytes in all age groups. In addition, IAIP co-localization with GFAP-positive astrocytes was more abundant in adults than in the developing brain. We conclude that IAIPs exhibit ubiquitous expression, and co-localize with neurons and astrocytes in the developing and adult human brain suggesting a potential role for IAIPs in development and endogenous neuroprotection.

Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain.

Hypoxic-ischemic brain injury is a leading cause of neurodevelopmental morbidities in preterm and full-term infants. Blood-brain barrier dysfunction represents an important component of perinatal hypoxic-ischemic brain injury. The extracellular matrix (ECM) is a vital component of the blood-brain barrier. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are important ECM components. They contribute to brain development, blood-brain barrier maintenance, and to regenerative and repair processes after hypoxic-ischemic brain injury. We hypothesized that ischemia at different durations of reperfusion affects the ECM protein composition of MMPs and TIMPs in the cerebral cortex of fetal sheep. Cerebral cortical samples were snap-frozen from sham control fetuses at 127 days of gestation and from fetuses after exposure to 30-min carotid occlusion and 4-, 24-, and 48-h of reperfusion. Protein expression of MMP-2, -8, -9, and -13 and TIMP-1, -2, -3, and -4 was measured by Western immunoblotting along with the gelatinolytic activity of MMP-2 and MMP-9 by zymography. The expression of MMP-8 was increased (Kruskal-Wallis, p = 0.04) in fetuses 48 h after ischemia. In contrast, changes were not observed in the protein expression of MMP-2, -9, or -13. The gelatinolytic activity of pro-MMP-2 was increased (ANOVA, p = 0.02, Tukey HSD, p = 0.05) 24 h after ischemia. TIMP-1 and -3 expression levels were also higher (TIMP-1, ANOVA, p = 0.003, Tukey HSD, p = 0.01; TIMP-3, ANOVA, p = 0.006, Tukey HSD, p = 0.01) 24 h after ischemia compared with both the sham controls and with fetuses exposed to 4 h of reperfusion. The changes in the expression of TIMP-1, -2, and -3 correlated with the changes in the MMP-8 and -13 protein expression. We speculate that regulation of MMP-8, MMP-13, and TIMPs contributes to ECM remodeling after is chemic-reperfusion injury in the fetal brain.

Neurological consequences of systemic inflammation in the premature neonate.

Despite substantial progress in neonatal care over the past two decades leading to improved survival of extremely premature infants, extreme prematurity continues to be associated with long term neurodevelopmental impairments. Cerebral white matter injury is the predominant form of insult in preterm brain leading to adverse neurological consequences. Such brain injury pattern and unfavorable neurologic sequelae is commonly encountered in premature infants exposed to systemic inflammatory states such as clinical or culture proven sepsis with or without evidence of meningitis, prolonged mechanical ventilation, bronchopulmonary dysplasia, necrotizing enterocolitis and chorioamnionitis. Underlying mechanisms may include cytokine mediated processes without direct entry of pathogens into the brain, developmental differences in immune response and complex neurovascular barrier system that play a critical role in regulating the cerebral response to various systemic inflammatory insults in premature infants. Understanding of these pathologic mechanisms and clinical correlates of such injury based on serum biomarkers or brain imaging findings on magnetic resonance imaging will pave way for future research and translational therapeutic opportunities for the developing brain.

Neutralizing anti-interleukin-1ß antibodies reduce ischemia-related interleukin-1ß transport across the blood-brain barrier in fetal sheep.

Hypoxic ischemic insults predispose to perinatal brain injury. Pro-inflammatory cytokines are important in the evolution of this injury. Interleukin-1ß (IL-1ß) is a key mediator of inflammatory responses and elevated IL-1ß levels in brain correlate with adverse neurodevelopmental outcomes after brain injury. Impaired blood-brain barrier (BBB) function represents an important component of hypoxic-ischemic brain injury in the fetus. In addition, ischemia-reperfusion increases cytokine transport across the BBB of the ovine fetus. Reducing pro-inflammatory cytokine entry into brain could represent a novel approach to attenuate ischemia-related brain injury. We hypothesized that infusions of neutralizing IL-1ß monoclonal antibody (mAb) reduce IL-1ß transport across the BBB after ischemia in the fetus. Fetal sheep were studied 24-h after 30-min of carotid artery occlusion. Fetuses were treated with placebo- or anti-IL-1ß mAb intravenously 15-min and 4-h after ischemia. Ovine IL-1ß protein expressed from IL-1ß pGEX-2T vectors in Escherichia coli (E. coli) BL-21 cells was produced, purified, and radiolabeled with 125I. BBB permeability was quantified using the blood-to-brain transfer constant (Ki) with 125I-radiolabeled-IL-1ß. Increases in anti-IL-1ß mAb were observed in the brain of the mAb-treated group (P<0.001). Blood-to-brain transport of 125I-IL-1ß was lower (P<0.04) across brain regions in the anti-IL-1ß mAb-treated than placebo-treated ischemic fetuses. Plasma 125I-IL-1ß counts were higher (P<0.001) in the anti-IL-1ß mAb- than placebo-treated ischemic fetuses. Systemic infusions of anti-IL-1ß mAb reduce IL-1ß transport across the BBB after ischemia in the ovine fetus. Our findings suggest that conditions associated with increases in systemic pro-inflammatory cytokines and neurodevelopmental impairment could benefit from an anti-cytokine therapeutic strategy.

Vectorial capacity and genetic diversity of Anopheles annularis (Diptera: Culicidae) mosquitoes in Odisha, India from 2009 to 2011.

Anopheles annularis is one of the major vectors of malaria in Odisha, India. The present study was undertaken to determine the vectorial capacity and assess the genetic diversity of An. annularis collected from different endemic regions of Odisha. Mosquitoes were collected from thirteen endemic districts using standard entomological collection methods from 2009 to 2011. Sibling species of An. annularis were identified by PCR-RFLP and sequencing of D3 region of 28S ribosomal DNA (rDNA) region. Plasmodium falciparum (Pf) sporozoite rate and human blood fed percentage (HBF) were estimated by multiplex PCR using Pf and human specific primers. Genetic diversity of An. annularis was estimated by ISSR markers. Out of 1647 An. annularis collected, 1353 (82.15%) were collected by mechanical aspirators and 294 (17.85%) by light trap. 49 (2.97%) were positive for human blood and 18 (1.09%) were positive for Pf sporozoite. PCR-RFLP and sequencing analyses detected only An annularis A in the study areas. Overall genetic differentiation among An. annularis populations was moderate (FST=0.048) and showed significant correlation between genetic distance and geographic distance (r=0.882; P<0.05). Angul population proved to be genetically unique and was highly divergent FST>0.110) from other populations, suggesting low gene flow between them. The study indicated that only An. annularis A was found in Odisha with potential vectorial capacity that can play a major role in malaria transmission. ISSR markers proved to be useful molecular tools to evaluate genetic variability in An. annularis populations.

Genetic characterization of E2 region of Chikungunya virus circulating in Odisha, Eastern India from 2010 to 2011.

Chikungunya virus (CHIKV) infection has caught attention yet again as it rages around the globe affecting millions of people. The virus caused epidemic outbreaks affecting more than 15,000 people in Odisha, Eastern India since 2010. In this study, complete genetic characterization of E2 gene of CHIKV circulating in Odisha from 2010 to 2011 was performed by virus isolation, RT-PCR, molecular phylogenetics and bioinformatics methods. Phylogenetic analyses revealed the circulation of Indian Ocean Lineage (IOL) strains of ECSA genotype of CHIKV in Odisha. Several mutations were detected in the E2 gene, viz. E2-R82G, E2-L210Q, E2-I211T, E2-V229I and E2-S375T which had various adaptive roles during the evolution of CHIKV. The CHIKV E2 peptide 57KTDDSHD6³ was predicted to be the most probable T-cell epitope and peptide 84FVRTSAPCT9² predicted to be the common T and B cell epitope having high antigenicity. The amino acid positions 356-379 and 365-385 were predicted to be transmembrane helical domains and indicated E2 protein anchorage in intracellular membranes for effective interaction with the host receptors. Positive selection pressure was observed in five specific sites, 210, 211, 318, 375, and 377 which were observed to be fixed advantageously in most viral isolates. Structural modeling revealed that E2 gene of CHIKV was composed of 3 domains and the major adaptive mutations were detected in domain B, which can modulate binding of CHIKV to host cells, while the transmembrane domain in domain C and the epitopes were located in domain A, which was found to be most conserved. This is the first report from Eastern India demonstrating a predictive approach to the genetic variations, epitopic regions and the transmembrane helices of the E2 region. The results of this study, combined with other published observations, will expand our knowledge about the E2 region of CHIKV which can be exploited to develop control measures against CHIKV.

Anopheles culicifacies sibling species in Odisha, eastern India: First appearance of Anopheles culicifacies E and its vectorial role in malaria transmission.

OBJECTIVE: To identify the Anopheles culicifacies sibling species complex and study their vectorial role in malaria endemic regions of Odisha. METHODS: Mosquitoes were collected from 6 malaria endemic districts using standard entomological collection methods. An. culicifacies sibling species were identified by multiplex polymerase chain reaction (PCR) using cytochrome oxidase subunit II (COII) region of mitochondrial DNA. Plasmodium falciparum (Pf) sporozoite rate and human blood fed percentage (HBF) were estimated by PCR using Pf- and human-specific primers. Sequencing and phylogenetic analysis were performed to confirm the type of sibling species of An. culicifacies found in Odisha. RESULTS: Multiplex PCR detected An. culicifacies sibling species A, B, C, D and E in the malaria endemic regions of Odisha. An. culicifacies E was detected for the first time in Odisha, which was further confirmed by molecular phylogenetics. Highest sporozoite rate and HBF percentage were observed in An. culicifacies E in comparison with other sibling species. An. culicifacies E collected from Nawarangapur, Nuapara and Keonjhar district showed high HBF percentage and sporozoite rates. CONCLUSION: An. culicifacies B was the most abundant species, followed by An. culicifacies C and E. High sporozoite rate and HBF of An. culicifacies E indicated that it plays an important role in malaria transmission in Odisha. Appropriate control measures against An. culicifacies E at an early stage are needed to prevent further malaria transmission in Odisha.

Molecular investigations of chikungunya virus during outbreaks in Orissa, Eastern India in 2010.

Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by mosquitoes of genus Aedes, mainly Aedes aegypti and Aedes albopictus. The resurgence of CHIKV in different parts of India is a point of major public health concern. In 2010, chikungunya outbreaks with high epidemic magnitude were recorded in coastal areas of Orissa, Eastern India, affecting more than 15,000 people coupled with severe arthralgia and prolonged morbidites. Detailed entomological, serological and molecular investigation of this unprecendented outbreak was carried out by collecting and studying 1359 mosquito samples belonging to A. albopictus, A. aegypti, A. vittatus, A. edwardsii and Culex species and 220 patients serum from the affected areas. In this study, CHIKV specific IgM capture-ELISA and reverse-transcription PCR (RT-PCR) were done to detect recent infection of CHIKV in serum samples and adult mosquitoes collected from the affected areas. The high maximum likelihood estimate (MLE) (15.2) in A. albopictus mosquitoes indicated that it was the principal vector involved in transmission of CHIKV in Orissa. Phylogenetic analysis revealed that the CHIKV strains involved in the outbreak belonged to the Indian Ocean Lineage (IOL) group within the East, Central and South African (ECSA) genotype. Genetic characterization of envelope glycoprotein (E1 and E2) genes revealed that all the CHIKV isolates from Orissa had the E1-A226V mutation that enhances viral dissemination and transmissibility by A. albopictus mosquitoes along with E2-L210Q and E2-I211T mutations, which play an epistatic role with E1-A226V mutation in adaptation of CHIKV to A. albopictus by increasing its midgut infectivity, thereby favoring its vectorial capacity. Our results showed the involvement of A. albopictus vector in the recent outbreaks in Orissa and circulation of IOL strains of ECSA genotype of CHIKV with E1-A226V, E2-L210Q and E2-I211T mutations in vectors and patients serum.

Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species.

OBJECTIVE: To develop a single-step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR. METHODS: Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single-step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas. RESULTS: A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus. CONCLUSIONS: The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic/epidemic areas where several species coexist.