Chemically induced alterations in maternal homeostasis and histology of conceptus: their etiologic significance in rat fetal anomalies

Possible relationships between maternal acid-base-electrolyte imbalance, histological changes in the maternal/extraembryonic tissues (decidua, placenta, membranes enclosing cavities), and fetal anomalies induced by maternotoxic doses of ethylene glycol, sodium salicylate, and cadmium chloride in rats were investigated. Acid-base-electrolyte, histologic and, teratologic studies were conducted concurrently with, as far as feasible, a similar protocol. Ethylene glycol caused 1) maternal homeostatic changes including metabolic acidosis and hyperosmolality, 2) extraembryonic lesions with degeneration of allantois and reduced villigenesis being more prevalent, and 3) materno-fetal effects such as decreases in fetal and maternal body weights, decreased maternal food intake, and fetal abnormalities (vertebral, rib, and sternebral defects). Few of these changes occurred when NaHCO3, an endogenous agent known to correct metabolic acidosis, was coadministered with ethylene glycol. Ethylene glycol-induced maternal metabolic acidosis, concurrent with hyperosmolality, was suspected to contribute toward reduction in villigenesis and fetal anomalies, including body weight reductions. Sodium salicylate induced the following: 1) mild maternal acidosis, hypokalemia, and hypophosphatemia with no significant change in pH; 2) maternal hemorrhage in extraembryonic cavities, papillary proliferation of the visceral yolk sac endoderm, and failure to form the chorioallantoic labyrinth; and 3) resorptions, hydrocephaly, rib defects, and fetal body weight reduction. Upon simultaneous treatment with sodium salicylate, NaHCO3 significantly reduced, and NH4Cl enhanced the incidence of the above histologic and teratologic effects, without significantly altering acid-base values. An etiologic association between the above salicylate-induced maternal and extraembryonic lesions and teratogenicity was likely. Cadmium chloride, whether administered by the intraperitoneal (ip) or intravenous (iv) route, caused 1) hydrocephaly, anophthalmia, vertebral and rib defects, reduction in fetal body weight, resorptions and maternal toxicity (acute peritonitis by the ip route only), and 2) extensive necrosis and hemorrhage in the decidua basalis, hemorrhage in the ectoplacental cone and around Reichert's membrane, and absence of chorioallantoic labyrinth. An etiologic relationship between these teratologic and histologic effects seemed probable, since both were dose-related. From the above studies, it was hypothesized that maternal factors--metabolic acidosis, hyperosmolality, hemorrhages in the ectoplacental cone, extraembryonic cavities, and around Reichert's membrane, and necrosis of decidua basalis--may have, directly or indirectly, reduced fetal nutrition and materno-embryonic gaseous exchange, which ultimately altered fetal development.     

Maternal toxicity: a possible etiological factor in embryo-fetal deaths and fetal malformations of rodent-rabbit species

Data from animal teratology studies were surveyed to determine whether embryo-fetal mortality and fetal malformations result from a primary action of the agent on the conceptus or if they are secondary to maternal toxicity--a consequence of administration with high dose levels of test chemicals. A fairly strong association between embryo-fetal mortality and maternal toxicity was revealed by analysis of data from hamsters, mice, rats, and rabbits in 234 studies of chemical and physical agents, of which 83 were conducted at both maternotoxic and nonmaternotoxic doses, 94 only at maternotoxic doses, and 49 at nonmaternotoxic doses. In the above studies, only nine chemicals (four each in hamsters and rabbits and one in rats) were reported to induce embryo-fetal deaths at apparently nonmaternotoxic doses. These findings tend to suggest a contributory role for maternal toxicity in the induction of embryo-fetal deaths. The previously reported hypothesis that certain fetal defects in mice may perhaps be caused by maternal toxicity was also found to be true in a review of data on hamsters, rats, and rabbits. Salient maternal toxicity-associated fetal malformations were exencephaly, encephalocele, micro- or anophalmia, and fused ribs in hamsters and defective (fused, missing, or extra) ribs, vertebrae, and sternebrae, ex-, an-, or microphthalmia, and cleft palate in rats and rabbits. These malformations occurred at low frequencies, generally with no readily apparent dose-response relationship. Presumptive evidence indicates that embryo-fetal deaths, and the above-mentioned fetal malformations in experimental animals, which in published literature are presently attributed to chemical induction for a large number of chemicals, may be a consequence of maternal toxicity per se.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Maternal toxicity--a possible factor in fetal malformations in mice

The assumption that major fetal malformations are indicative of a chemical's teratogenic potential was not supported by a literature review of teratology studies conducted in mice. In these studies, dose levels of test agents that manifested maternal toxicity as suggested by reduction in dam's body weight, clinical signs of toxicity, or deaths, also invariably caused reduction in fetal body weight, increased resorptions, and rarely fetal deaths. In several such studies, conducted with maternotoxic doses of structurally unrelated test agents, a consistent pattern of fetal defects was discovered. These defects included exencephaly, open eyes, hemivertebrae, fused arches or centra of lumbar or thoracic vertebrae, fused, missing or supernumerary ribs, and fused or scrambled sternebrae. These defects were absent at drug dosages that were distinctly nontoxic for the mother. In a few studies conducted at two or more maternally toxic doses, the degree and severity of maternal toxicity showed a positive correlation with the incidence and severity of above fetal defects. It is hypothesized that maternal toxicity, on its own, may have an etiologic role in these fetal defects.     

Fine structural observations of the cotyledons in germinating seeds ofPelargonium XHortorum Bailey: With normal and mutant plastids

Summary The fine structural changes in cotyledon cells of germinatingPelargonium seeds are studied on the first to fifth day of seedling emergence. Initially there is a rapid change in the cell fine structure, marked most conspicuously by the progressive liberation of the lipid and protein food reserves and the formation of an extensive thylakoid system within the plastids, but gradually the cells start to senesce. The subcellular changes in mutant cells are similar except that the plastids lack the usual thylakoid system and develop only deranged prolamellar bodies. They may store starch and they possess plastoglobuli, but seem not to contain plastid ribosomes. In rare mixed cells normal and mutant plastids remain quite distinct.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.     

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Background

Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/Principal Findings

We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that “rBCG prime - DNA boost” strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.