Free-living, Amphizoic and Opportunistic Amebas

Amebas belonging to the genera Naegleria, Acanthamoeba and Balamuthia are free-living, amphizoic and opportunistic protozoa that are ubiquitous in nature. These amebas are found in soil, water and air samples from all over the world. Human infection due to these amebas involving brain, skin, lung and eyes has increased significantly during the last 10 years. The epidemiology, immunology, protozoology, pathology, and clinical features of the infections produced by these protozoa differ strikingly. Infection by the pathogenic Naegleria fowleri is acquired by exposure to polluted water in ponds, swimming pools and man-made lakes. Raised temperatures during the hot summer months or warm water from power plants facilitate the growth of N. fowleri. N. fowleri is a thermophilic ameba that grows well in tropical and subtropical climates. The CNS infection, called Primary Amebic Meningoencephalitis (PAM), produced by N. fowleri is characterized by an acute fulminant meningoencephalitis leading to death 3–7 days after exposure. Victims are healthy, young individuals with a history of recent water-related sport activities. The portal of entry is the olfactory neuroepithelium. The pathologic changes are an acute hemorrhagic necrotizing meningoencephalitis with modest purulent exudate, mainly at the base of the brain, brainstem and cerebellum. Trophozoites can be seen within the CNS lesions located mainly around blood vessels. Thus far 179 cases have been reported; 81 in the USA alone. Balamuthia mandrillaris and several species of Acanthamoeba are pathogenic “opportunistic” free-living amebas which cause Granulomatous Amebic Encephalitis (GAE) in humans and animals. GAE is an infection, usually seen in debilitated, malnourished individuals, in patients undergoing immunosuppressive therapy for organ transplants and in Acquired Immunodeficiency Syndrome (AIDS). The granulomatous component is negligible, particularly in immunocompromised individuals. Pathologically these amebas produce a patchy, chronic or subacute granulomatous encephalitis with the presence of trophozoites and cysts. The portal of entry is probably through the respiratory tract or an ulceration of the skin reaching the CNS by hematogenous spread. As of October 1, 1996, 166 cases 1103 due to Acanthamoeba and 63 due to Balamothial of GAE have been reported from around the world. Of these 103 cases due to Acanthamoeba (72 have been reported in the USA alone, > 50 in AIDS). It is well known that several species of Acanthamoeba can also produce, chronic sight threatening ulceration of the cornea called Acanthamoeba keratitis (AK), mostly in contact lens wearers or in individuals with minor corneal abrasions. Hundreds of cases of AK have been documented world wide.     

Caspase-dependent and -independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis

BACKGROUND: Germ cell elimination and sperm DNA fragmentationin men with primary testiculopathies involve apoptosis-relatedprocesses whose mechanisms are poorly understood. This studyexamines the participation of typical (caspase-dependent) andatypical (caspase-independent) pathways in these processes.METHODS: Caspase activity and DNA fragmentation were evaluatedin Sertoli and germ cells from 63 men with non-obstructive azoospermiaand with different histological diagnoses who were undergoingtesticular biopsy for an assisted reproduction attempt. In eightof these men, phosphatidylserine externalization was also examined.RESULTS: The percentage of Sertoli cells showing caspase activityand DNA fragmentation was low and uniform in all diagnoses.In germ cells that remained tightly associated with Sertolicells despite vigorous mechanical treatment, the incidence ofboth caspase activity and DNA fragmentation was high, particularlyin men with maturation arrest. In Sertoli cell-free germ cells,high incidence of DNA fragmentation contrasted with low incidenceof caspase activity and phosphatidylserine externalization.CONCLUSIONS: In men with primary testicular failure, apoptosisof Sertoli cells is insignificant. Some germ cells undergo caspase-dependentapoptosis, show phosphatidylserine externalization and are tightlyassociated with Sertoli cells. Other germ cells show caspase-independentDNA fragmentation, do not externalize phosphatidylserine andlack a tight association with Sertoli cells.     

Cross-linking of surface IgM or IgD causes differential biological effects in spite of overlap in tyrosine (de)phosphorylation profile.

Although displaying similar amounts of surface IgM and IgD, ECH 408-1 cells only succumb to apoptosis after cross-linking of IgM (not IgD), suggesting that different signaling pathways couple to both receptors. Immunoprecipitation studies revealed the presence of several proteins selectively associated with IgM and IgD, thus ruling out that the lack of inhibitory signaling mediated by IgD might be due to membrane expression in the absence of associated proteins belonging to the B cell receptor complex. 32P metabolic labeling and immunoprecipitation studies demonstrated that IgM and IgD are associated with phosphoproteins of 32-33 kDa in an isotype-specific fashion. Kinetic analyses of tyrosine kinase activity showed that cross-linking of surface IgM or IgD resulted in the rapid (1-3 min) phosphorylation of several protein substrates on tyrosine residues, followed by a dephosphorylation step. Isotype-specific changes of the phosphorylation status specifically affected molecules in the 32-33 kDa range, i.e. IgM (not IgD) cross-linking affected a approximately 32-kDa protein, whereas IgD (not IgM) cross-linking induced phosphorylation of a protein exhibiting a slightly lower mobility (33 kDa). These results suggest that isotype-specific immunoglobulin-associated molecules could be involved in the second messenger cascade leading to different biological effects upon IgM and IgD cross-linking.     

Implication of biofilm formation in the persistence of urinary tract infection caused by uropathogenic Escherichia coli

Escherichia coli is the most frequent microorganism involved in urinary tract infection (UTI). Acute UTI caused by uropathogenic E. coli (UPEC) can lead to recurrent infection, which can be defined as either re-infection or relapse. E. coli strains causing relapse (n = 27) and re-infection (n = 53) were analysed. In-vitro production of biofilm, yersiniabactin and aerobactin was significantly more frequent among strains causing relapse. Biofilm assays may be helpful in selecting patients who require a therapeutic approach to eradicate persistent biofilm-forming E. coli strains and prevent subsequent relapses.     

Microdissection genotyping of mixed glial and primitive neuroectodermal central nervous system neoplasm

A 22-year-old man with previous radiation treatment for childhood astrocytoma underwent resection of a right parietooccipital lesion. Histopathology revealed a malignant neoplasm with areas of astrocytic and primitive neuroectodermal components. To resolve the relationship and cellular origin, representative tissue was microdissected from several targets, obtaining a balanced mixture of each element. Nonneoplastic brain parenchyma was separately microdissected to determine polymorphic marker informativeness and to serve as an internal negative control. Despite the relatively small quantity of tissue removed for each microdissection target, sufficient material was available for reliable, balanced, polymerase chain reaction-format genotyping encompassing a panel of tumor suppressor genes and genetic loci associated with these forms of neoplasia. The findings revealed distinct discordant genotypic profiles for each of the neoplastic components. The efficacy of the approach used for molecular analysis of this complex neoplasm and the implication of the genotypic findings are discussed.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

The integrity of sperm DNA is crucial for the maintenance of genetic health. A major source of damage is reactive oxygen species (ROS) generation; therefore, antioxidants may afford protection to sperm DNA. The objectives of the study were, first, to measure the effects of antioxidant supplementation in vitro on endogenous DNA damage in spermatozoa using the single cell gel electrophoresis (comet) assay and, second, to assess the effect of antioxidant supplementation given prior to X-ray irradiation on induced DNA damage. Spermatozoa from 150 patients were prepared by Percoll centrifugation in the presence of ascorbic acid (300, 600 microM), alpha tocopherol (30, 60 microM), urate (200, 400 microM), or acetyl cysteine (5, 10 microM). DNA damage was induced by 30 Gy X-irradiation. DNA strand breakage was measured using the comet assay. Sperm DNA was protected from DNA damage by ascorbic acid (600 microM), alpha tocopherol (30 and 60 microM) and urate (400 microM). These antioxidants provided protection from subsequent DNA damage by X-ray irradiation. In contrast, acetyl cysteine or ascorbate and alpha tocopherol together induced further DNA damage. Supplementation in vitro with the antioxidants ascorbate, urate and alpha tocopherol separately has beneficial effects for sperm DNA integrity.      

Phenotype delineation of ZNF462 related syndrome

Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.     

Purification and properties of rabbit alveolar macrophage lysozyme.

Lysozyme was isolated from Bacillus Calmette-Guerin-elicited rabbit alveolar macrophages by acid extraction and purified to homogeneity by a single-column procedure. Yields of the purified enzyme averaged between 20 and 30 mg per rabbit, values far in excess of those obtained with previously published methods. Rabbit lysozyme has a molecular weight of 14,300 and exhibits optimal lytic activity against Micrococcus lysodeikticus at an ionic strength of 0.04, pH 6.5. Our results indicate that lysozyme and other granule components can be fractionated from elicited alveolar macrophages by using simple techniques, suggesting methods for the bulk purification of lysosomal constituents.     

Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration

The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-gamma) in the central nervous system (CNS). However, IFN-gamma-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-gamma-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-gamma-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-gamma-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E(2) (PGE(2)) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE(2) and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE(2) secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.