Impact of COVID-19 during pregnancy on placental pathology, maternal and neonatal outcome - A cross-sectional study on anemic term pregnant women from a tertiary care hospital in southern India.

Background: SARS-CoV-2 infection during pregnancy may cause adverse maternal, neonatal and placental outcomes. While tissue hypoxia is often reported in COVID-19 patients, pregnant women with anemia are suspected to be more prone to placental hypoxia-related injuries. Methods: This hospital-based cross-sectional study was conducted between August-November 2021, during COVID-19 second wave in India. Term pregnant women (N=212) admitted to hospital for delivery were enrolled consecutively. Since hospital admission mandated negative RT-PCR test for SARS-CoV-2 virus, none had active infection. Data on socio-demography, COVID-19 history, maternal, obstetric, and neonatal outcomes were recorded. Pre-delivery maternal and post-delivery cord blood samples were tested for hematological parameters and SARS-CoV-2 IgG. Placentae were studied for histology. Results: Of 212 women, 122 (58%) were seropositive for SARS-CoV-2 IgG, but none reported COVID-19 history; 134 (63.2%) were anemic. In seropositive women, hemoglobin (p=0.04), total WBC (p=0.009), lymphocytes (p=0.005) and neutrophils (p=0.02) were significantly higher, while ferritin was high, but not significant and neutrophils to lymphocytes (p=0.12) and platelets to lymphocytes ratios (p=0.03) were lower. Neonatal outcomes were similar. All RBC parameters and serum ferritin were significantly lower in anemic mothers but not in cord blood, except RDW that was significantly higher in both, maternal (p=0.007) and cord (p=0.008) blood from seropositive anemic group compared to other groups. Placental histology showed significant increase in villous hypervascularity (p=0.000), dilated villous capillaries (p=0.000), and syncytiotrophoblasts (p=0.02) in seropositive group, typically suggesting placental hypoxia. Maternal anemia was not associated with any histological parameters. Univariate and multivariate logistic regression analyses of placental histopathological adverse outcomes showed strong association with SARS-CoV-2 seropositivity but not with maternal anemia. When adjusted for several covariates, including anemia, SARS-CoV-2 seropositivity emerged as independent risk factor for severe chorangiosis (AOR 8.74, 95% CI 3.51-21.76, p<0.000), dilated blood vessels (AOR 12.74, 95% CI 5.46-29.75, p<0.000), syncytiotrophoblasts (AOR 2.86, 95% CI 1.36-5.99, p=0.005) and villus agglutination (AOR 9.27, 95% CI 3.68-23.32, p<0.000). Conclusion: Asymptomatic COVID-19 during pregnancy seemed to be associated with various abnormal placental histopathologic changes related to placental hypoxia independent of maternal anemia status. Our data supports an independent role of SARS-CoV-2 in causing placental hypoxia in pregnant women.

SARS-CoV-2 seroprevalence in the city of Hyderabad, India in early 2021.

Background: COVID-19 emerged as a global pandemic in 2020, spreading rapidly to most parts of the world. The proportion of infected individuals in a population can be reliably estimated via serosurveillance, making it a valuable tool for planning control measures. Our serosurvey study aimed to investigate SARS-CoV-2 seroprevalence in the urban population of Hyderabad at the end of the first wave of infections. Methods: This cross-sectional survey, conducted in January 2021 and including males and females aged 10 years and above, used multi-stage random sampling. 9363 samples were collected from 30 wards distributed over six zones of Hyderabad, and tested for antibodies against SARS-CoV-2 nucleocapsid antigen. Results: Overall seropositivity was 54.2%, ranging from 50% to 60% in most wards. Highest exposure appeared to be among those aged 30-39 and 50-59 years, with women showing greater seropositivity. Seropositivity increased with family size, with only marginal differences among people with varying levels of education. Seroprevalence was significantly lower among smokers. Only 11% of the survey subjects reported any COVID-19 symptoms, while 17% had appeared for COVID-19 testing. Conclusion: Over half the city's population was infected within a year of onset of the pandemic. However, ∼ 46% of people remained susceptible, contributing to subsequent waves of infection.

Oral Toxicity Study for Salmonella Killing Lytic Bacteriophage NINP13076 in BALB/c Mice and Its Effect on Probiotic Microbiota.

Viruses that infect bacteria are emerging as attractive biocontrol agents and biopreservatives for foods. Since these bacteriophages kill the target pathogens by lysis and are also consumed along with food, it is essential to evaluate their collateral toxicity on the probiotic gut microbiota. In this study, we examined the acute oral toxicity of a Salmonella phage isolated from sewage in mice. Acute oral administration of the Salmonella phage for five consecutive days did not show any significant pathological changes in the vital organs like lung, kidneys, heart, liver, and intestine. In addition, growth of typical probiotic microbiota remained unaffected even after incubation up to 24 h with the Salmonella phage. The results of this study clearly showed that oral administration of the lytic Salmonella phage did not have any significant adverse effects on the animals, may not harm the probiotic gut microbiota, and are likely to be safe for use in food preservation.

Impact of maternal iron deficiency anaemia on the expression of the newly discovered multi-copper ferroxidase, Zyklopen, in term placentas.

In the present study, we investigated the effect of maternal iron deficiency anaemia (IDA) on expression of the newly discovered iron transporter, Zyklopen in term placenta, in 200 pregnant women. Placental expression of Zyklopen was studied by mRNA analysis and immunohistochemistry for the protein. In addition neonatal anthropometric parameters were also analysed. 58.8% of 200 subjects were anaemic. Both Zyklopen mRNA as well as protein expression in the placenta showed a statistically significant increase with increasing severity of anaemia. Although all the neonatal anthropometric parameters were lower in newborns of anaemic mothers, none showed any statistical significance. Zp mRNA levels did not show any significant correlation with newborn and placental parameters (except newborn skinfold thickness and head circumference). Similar to mRNA expression, Zp IHC expression correlated positively, albiet non-significantly, with newborn length and Hb levels, the correlation was however negative with birth weight, head circumference, mid-arm circumference unlike the mRNA expression, where it positively correlated with the above parameters. Our study for the first time demonstrated a definite increase in expression of Zyklopen at both mRNA and protein levels in term placenta, in maternal IDA.IMPACT STATEMENTWhat is already known on this subject? Iron deficiency anaemia (IDA) in a pregnant mother can lead to anaemia in the developing foetus; which is frequently observed to be of lesser severity than that in the mother. Recently a copper-containing oxidase called Zyklopen was discovered which was involved in iron efflux in BeWo cells. The gene encoding Zyklopen has been identified with a putative C-terminal membrane-spanning sequence and high sequence identitical to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate multicopper ferroxidase (MCF). Protein expression of this new MCF was observed in multiple diverse mouse tissues, including placenta and mammary gland.What do the results of this study add? Zyklopen protein immunohistochemical expression showed a statistically significant increase with increasing severity of anaemia. Similarly, placental mRNA expression of the Zyklopen gene was observed to be higher in anaemic mothers when compared to non-anaemic mothers. Our study for the first time demonstrated a definite increase in expression of Zyklopen at both protein and mRNA levels in term placenta, in maternal IDA.What are the implications of these findings for clinical practice and/or further research? This study will help us to understand better, the increased potential for influx of iron from mother to foetus in the condition of maternal iron deficiency. This study will help to determine how placental iron transport proteins can be regulated in response to maternal and neonatal iron status and will further our existing knowledge on relationships between maternal and neonatal iron status and mechanisms by which placental iron transport is modified in relation to these parameters.

An innovative method of nonclinical efficacy and toxicological evaluation of recombinant Salmonella typhi Ty21a expressing HPV16 and 18 L1 proteins.

Advancement in technology led to development of live attenuated Salmonella typhi Ty21a as enteric vector for expression of foreign proteins. Such vector platform is inevitable for development of vaccine candidate against human papilloma virus (HPV), the etiological agent of cervical cancer with high prevalence in developing nations. The high risk HPVs like type 16 and 18 contributes to 70% of cervical cancer, hence Indian Immunologicals Limited (IIL), Hyderabad, India developed a recombinant HPV vaccine by introducing HPV 16 and 18 L1 protein coding genes into attenuated S. typhi Ty21a vector. Being a genetically engineered enteric vector vaccine, it would be less expensive, with an ease of oral administration, instead of injectable that needs trained personale, is an added advantage for low socioeconomic setup compared to existing HPV vaccines. Establishing the nonclinical efficacy and safety/toxicity as per the national/international regulatory guidelines has become major constrain for such recombinant S. typhi HPV (rSt.HPV) vaccine. Since, the intended clinical mode of rSt.HPV is through oral route, whereas the live attenuated S. typhi Ty21a doesn't colonize in gut of laboratory animals to be used for nonclinical experiments. Hence, an alternate and unconventional method of 'intranasal drug testing', was followed for nonclinical efficacy and toxicity evaluations. An array of parameters specified by regulatory agencies were investigated in mice, rat and rabbits administered with rSt.HPV through, intra-peritoneal, intranasal and oral routes, the intended clinical route.•Current unconventional and innovative nonclinical testing procedures helps in exploring the alternate methods by pharmacologist/toxicologist.•Ultimately, such new drugs developed through technology must serve the humankind justifying the guidelines of regulatory agencies.

Pharmacological properties of durva swaras (Cynodon dactylon L. Pers.) in an ovariectomised rat model mimicking chronic menopausal syndrome.

Hormonal replacement therapy (HRT), as the first-line management of chronic menopausal syndrome (CMS) in women, has limited application due to adverse effects. We aimed to evaluate the therapeutic potential of a herbal alternative (HALT), durva swaras (DS) of Cynodon dactylon L. Pers., in a CMS rat model. Female Sprague-Dawley rats were subjected to Sham and ovariectomy (OVX) surgery. OVX rats received either 0.11 mg/kg oestrogen as a positive treatment control or 1 (DS1), 2 (DS2), and 4 (DS3) g/kg DS for 160 days. Vaginal smear tests indicated the menopausal status. Routine clinical examinations, weekly body weights (BW), serum calcium, proinflammatory cytokines, and reproductive hormones levels were monitored. Clinical chemistry, body composition, bone mineral density (BMD), uterotrophic response, bone morphometry, and histopathology of major organs were evaluated. BW of OVX rats increased by 18-25% compared to Sham. Total fat and fat percentage were significantly elevated in the oestrogen group compared to DS2, DS3, and OVX group. DS treatment groups showed the levels of TNF- α was slightly reduced, while IL-1ß and IL-6 levels were significantly reduced (P < 0.05) compared to the oestrogen treated group. DS treatment restored serum calcium levels, while BMD, bone quality, osteoblast/osteoclast ratio, and collagen levels improved in both DS and oestrogen treatment groups. The uterotrophic assay demonstrated non-oestrogenic activity of DS. Endometrial hyperplastic change was observed in oestrogen-treated rats. The preclinical non-oestrogenic activity of DS has therapeutic potential in CMS through anti-inflammatory and osteo-protective effects. Further clinical research into DS, as a viable HALT to HRT, is required.

Toxicological data of rats and rabbits: Sub-chronic testing of recombinant HPV vaccine through oral and intranasal routes.

Current article illustrates the data of body weight, biochemical, haematological profile, and organ weights of rats and rabbits administered with recombinant human papilloma virus (HPV) vaccine, along with genotoxicity effect. The data was collected from nonclinical safety/toxicity and immune response evaluations of recombinant Salmonella typhi expressing the HPV 16 and 18 L1 proteins as vaccine. The intended clinical route of vaccine administration is through oral route, whereas it is established fact that attenuated S. typhi could not colonize in laboratory animals. In view of this it is challenging to undertake the nonclinical safety/toxicity evaluations following the regulatory guidelines. Hence sub chronic safety/toxicity testing was carried out in rat and rabbits by administration of HPV vaccine through oral (intended clinical route) and innovative intranasal routes. The prophylactic dose derived from adult human clinical dose (2 × 109CFU/70 kg) was administered to SD rats (PD: 0.18 × 109CFU/kg) and New Zealand White (NZW) rabbits (PD: 0.09 × 109CFU/kg) through oral and intranasal routes. Similarly, average dose (AD:5xPD) was administered to rats (AD:0.9 × 109CFU/kg) and rabbits (AD: 0.45 × 109CFU/kg) through intranasal route only. The repeated doses were administered on 3rd and 5th days of post-exposure of 1st dose through specified routes and test compound effects in relation with time of exposure was assessed by euthanizing animals and data collection at different time points i.e. 15th (25% of animals), 29th (25% of animals) and 93rd days (50% of animals) of post-exposure of 1st dose. The retro-orbital plexus blood was collected before euthanizing animals to unveil the biochemical and haematological profile. The data on genotoxicity effect of test compound, if any, was obtained by assessing the bone-marrow micronucleus assay. The immune response and allergenicity in terms of specific IgG and IgE levels against HPV 16 and 18 L1 proteins were determined in mice. The raw data of various parameters collected at different time points were compiled and computed according to the groups. The haematological profile and organ weights data can be used as reference data for SD rats and NZW rabbits for future studies.

Nonclinical safety evaluation of oral recombinant anti-human papilloma virus vaccine (RHPV 16 & 18): Regulatory toxicology studies in mice, rats and rabbits - An innovative approach.

AIM: The human papilloma virus (HPV) type 16 and 18 causes nearly 70% of uterine cervical cancers. Oral administration of live Salmonella typhi Ty21a, expressing major capsid proteins (L1) of HPV 16 and 18 is a potential choice for immunization in adolescent girls under low resource settings. Present study aimed to assess the nonclinical safety of recombinant S. typhi expressing HPV 16 and 18 (rStHPV) proteins. METHODOLOGY: The acute toxicity of rStHPV was tested by intranasal single dose administration, of 10 and 50 folds higher than clinical prophylactic dose, in mice and rat followed by monitoring their survival for 14 days. Sub-chronic toxicity was evaluated in rats and rabbits with prophylactic and 5 times (average) to clinical prophylactic dosages on scheduled days (1st, 3rd & 5th day) through oral and intranasal routes. The immune/allergic response of rStHPV was assessed in mice through intranasal and intra-peritoneal routes. Experimental animals were daily monitored for live phase, and clinical chemistry, haematology, immunotoxicology, immunogenic response and histopathological examination of vital organs on 15th, 29th and 93rd days. RESULTS: No abnormal changes were noticed in live phase activity, clinical chemistry and haematology profile. The gross necropsy, organ weights and histopathology were found to be normal. No immunotoxicity was recorded as evaluated by tier I tests. Allergic immune response, as evaluated with IgE levels was also negative irrespective of test routes. On the other hand, a significant (P < 0.01) increase of anti-HPV IgG levels was noted in mice exposed through intranasal route. Though the pre-terminal mortality was noted in mice (6-15%), rats (10%) and rabbits (15%), the autopsy revealed no signs of toxicity related to rStHPV, as the changes neither significant nor dose dependent; and even noted in vehicle control also. CONCLUSION: The study results suggested 'no observable adverse effects' of rStHPV even at higher dosages (5, 10 & 50 folds) than intended clinical dose. A significant increase of anti-HPV specific IgG suggests the immunogenicity of vaccine. The innovative approach of current study is nonclinical toxicology evaluation of vaccine through intra-nasal route, an alternate route apart from stipulated regulatory guidelines.

Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD).

BACKGROUND CONTEXT: One of the common causes of low back pain is intervertebral disc degeneration. The pathophysiology of disc degeneration involves apoptosis of nucleus pulposes cells and degradation of extra cellular matrix (ECM). Caspase 3 plays a central role in apoptosis and the ADAMTS5 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 5) gene plays a critical role in ECM degradation. Hence, we hypothesized that if one can silence these two genes, both apoptosis and ECM degradation can be prevented, thereby preventing the progression and even reverse disc degeneration. PURPOSE: The purpose of this study is to demonstrate the regenerative potential of small interfering RNA (siRNA) designed against Caspase 3 and ADAMTS5 genes in an in vitro and animal model of disc degeneration. STUDY DESIGN: In vitro study followed by in vivo study in a rabbit model. METHODS: In vitro studies were done using the human hepatocellular carcinoma (Hep G2) cell line for validating the efficacy of liposomal siRNA in controlling the expression of genes (Caspase 3 and ADAMTS5). Later, siRNA's validation was done in a rabbit annular punctured model by administering siRNA's individually (Caspase 3 and ADAMTS5) and in combination Caspase3-ADAMTS5) for assessing their synergistic effect in down regulating the gene expression in the degenerative discs. Annular punctured intervertebral discs of the rabbit were injected with siRNA formulations (single and dual) and phosphate buffer saline, one week after initial puncture. Magnetic resonance imaging (MRI) scans were done before and after siRNA treatment (1, 4 and 8 weeks) for assessing the progression of disc degeneration. The histopathology and real time polymerase chain reaction (RT-PCR) studies were done for evaluating their efficacy. We did not receive any funding for conducting the study, and we do not have a conflict of interest with any researchers or scientific groups. RESULTS: The observations made from both in vitro and in vivo studies indicate the beneficial effects of siRNA formulation in down regulating the expression of Caspase 3 and ADAMTS5 genes. The MRI and histopathological evaluation showed that the disc degeneration was progressive in phosphate buffer saline and AT5-siRNA injected discs but the discs that received Caspase 3-siRNA and dual siRNA (Cas3-AT5-siRNA) formulation showed signs of recovery and regeneration 4 and 8 weeks after injection. The efficacy of siRNA designed against Cas3 and AT5 was also assessed in both in vitro and in vivo experiments by using RT-PCR analysis and the results showed downregulation of Caspase 3 gene in Caspase 3-siRNA group, but there was no significant downregulation of ADAMTS5 gene in ADAMTS5-siRNA group (ie, indicated by fold change). Synergistic effect was observed in the group that received dual siRNA (Cas3-AT5 siRNA) formulation. CONCLUSIONS: This experiment suggests that intervention by siRNA treatment significantly reduced the extent of apoptosis in the discs. CLINICAL SIGNIFICANCE: Delivery of siRNA directly into spinal discs has a potential in treating disc degeneration nonsurgically.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is frequently upregulated in prostate cancer, and its overexpression conveys tumor growth and angiogenesis by metabolizing asymmetric dimethylarginine (ADMA).

Tissue microarray analysis confirmed higher dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression in prostate cancer (PCa) compared to benign and normal prostate tissues. DDAH1 regulates nitric oxide (NO) production by degrading endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA). This study examined whether DDAH1 has any physiological role in PCa progression. Using overexpression of DDAH1 in PCa (PC3 and LNCaP) cell lines, we found that DDAH1 promotes cell proliferation, migration and invasion by lowering ADMA levels, as well as increasing NO production. VEGF, HIF-1α and iNOS were upregulated in DDAH1 expressing cells as result of elevated NO. DDAH1 increased secretion of pro-angiogenic signals bFGF and IL-8, into conditioned media. Treatment of DDAH1-positive PCa cells with NOS inhibitors (L-NAME and 1400 W) attenuated DDAH1 activity to promote cell growth. Xenografts derived from these cells grew significantly faster (> twofold) than those derived from control cells. Proliferation rate of cells stably expressing mutant DDAH1 was same as control cells unlike wild-type DDAH1-positive PCa cells. Xenograft tumors derived from mutant-positive cells did not differ from control tumors. VEGF, HIF-1α and iNOS expression did not differ in DDAH1 mutant-positive tumors compared to control tumors, but was upregulated in wild-type DDAH1 overexpressing tumors. Furthermore, CD31 immunostaining on xenograft tissues demonstrated that DDAH1 tumors had high endothelial content than mutant DDAH1 tumors. These data suggest that DDAH1 is an important mediator of PCa progression and NO/DDAH pathway needs to be considered in developing therapeutic strategies targeted at PCa.

Combination of spices and herbal extract restores macrophage foam cell migration and abrogates the athero-inflammatory signalling cascade of atherogenesis.

The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, information about possible treatments for this condition is lacking. Here, we hypothesized that combining the polyphenol-rich fractions (PHC) of commonly consumed spices (Allium sativum L (Liliaceae), Zingiber officinale R (Zingiberaceae), Curcuma longa L (Zingiberaceae)) and herbs (Terminalia arjuna (R) W & A (Combretaceae) and Cyperus rotundus L (Cyperaceae)) prevents foam cell formation and atherogenesis. Using an in vitro foam cell formation assay, we found that PHC significantly inhibited lipid-laden macrophage foam cell formation compared to the depleted polyphenol fraction of PHC (F-PHC). We further observed that PHC attenuated the LDL and LPS induced CD36, p-FAK and PPAR-γ protein expression in macrophages and increased their migration. NK-κB-DNA interaction, TNF-α, ROS generation, and MMP9 and MMP2 protein expression were suppressed in PHC-treated macrophages. The anti-atherosclerotic activity of PHC was investigated in a high fat- and cholesterol-fed rabbit model. The inhibition of foam cell deposition within the aortic intima and atheroma formation confirmed the atheroprotective activity of PHC. Therefore, we conclude that the armoury of polyphenols in PHC attenuates the CD36 signalling cascade-mediated foam cell formation, enhances the migration of these cells and prevents atherogenesis.

Effect of cinnamon and its procyanidin-B2 enriched fraction on diabetic nephropathy in rats.

Non-enzymatic protein glycation and resultant accumulation of advanced glycation endproducts (AGE) are implicated in the pathogenesis of diabetic complications including diabetic nephropathy (DN). It is considered that antiglycating agents offer protection against AGE mediated pathologies including DN. Earlier we characterized procyanidin-B2 (PCB2) as the active component from cinnamon (Cinnamomum zeylanicum) that inhibits AGE formation in vitro. In this study, we have investigated the potential of PCB2-enriched fraction of cinnamon to prevent in vivo accumulation of AGE and to ameliorate renal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed with either 3% cinnamon or 0.002% PCB2-fraction in diet for 12weeks. Biochemical analysis of blood and urine was performed at the end of experiment. Evaluation of glomerular markers that serve as indicators of renal function was done by immunohistochemistry, immunoblotting and qRT-PCR. Supplementation of diabetic rats with cinnamon and PCB2-fraction prevented glycation mediated RBC-IgG cross-links and HbA1c accumulation in diabetes rats. Cinnamon and PCB2-fraction also inhibited the accumulation of N-carboxy methyl lysine (CML), a prominent AGE in diabetic kidney. Interestingly, cinnamon and its PCB2-fraction prevented the AGE mediated loss of expression of glomerular podocyte proteins; nephrin and podocin. Inhibition of AGE by cinnamon and PCB2-fraction ameliorated the diabetes mediated renal malfunction in rats as evidenced by reduced urinary albumin and creatinine. In conclusion, PCB2 from cinnamon inhibited AGE accumulation in diabetic rat kidney and ameliorated AGE mediated pathogenesis of DN.