Integrating central nervous system metagenomics and host response for diagnosis of tuberculosis meningitis and its mimics.

The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.

Autoradiography of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): uptake in the monoaminergic pathways and in melanin containing tissues.

A recently discovered neurotoxic compound, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, has been found to cause a parkinsonian-like syndrome in man and monkey, but not in laboratory animals such as rat, mouse and guinea pig. MPTP seems to selectively destroy the melanin containing dopaminergic cells in pars compacta of substantia nigra. Lower mammalian species do not have melanin in these cells, which indicates that the presence of neuromelanin may be of importance for the development of MPTP-induced lesions. By means of whole-body autoradiography of 3H-MPTP in mice, accumulation and retention was observed in the dopaminergic pathways, in locus caeruleus and in structures in the medulla oblongata and spinal cord. A high uptake was also seen in melanin-containing tissues such as in the eyes of pigmented mice. MPTP has earlier been found to have high affinity in vitro for dopamine melanin, which is similar to the pigment in substantia nigra. The typical features of the MPTP-induced neurotoxicity with destruction of pigmented nerve cells and development of parkinsonism may be to to accumulation and retention of MPTP and its metabolites in these cells. In species with pigmented nerve cells, such as man and monkey, the accumulation may be much more pronounced because of the melanin affinity of MPTP and its metabolites.

Insulin receptors on pancreatic acinar cells in guinea pigs.

The characteristics of interaction of insulin with specific receptors on exocrine pancreatic cells of the guinea pig have been studied. Insulins from different species as well as certain insulin analogs were found to have affinities to receptors on pancreatic acinar cells which are similar to what have been described for insulin receptors in other organs of different mammalian species. Binding was rapid and reversible at 37 C but dissociation was markedly slower at 12 C. Clear indications of negative cooperativity between binding sites were not seen. Bovine and chicken insulin bound with approximately a 100-fold higher affinity to guinea pig insulin receptors than guinea pig insulin itself. The number of insulin receptors per acinar cell were comparable with what has been described for other mammalian cells. Part of cell-associated insulin was internalized. After 60 min of incubation the major part of radioactivity in the incubation medium as well as in cells appeared as intact [125I] iodoinsulin on a Sephadex G-50 column and less than 12% of radioactivity was eluted as breakdown products together with Na 125I.

Melanin affinity of manganese.

The melanin affinity of manganese was studied in vitro using melanin isolated from beef eyes or human hair, and synthetic dopamine melanin, which is known to be structurally similar to the melanin present in the pigmented nerve cells in the human substantia nigra. In addition, the uptake of manganese in melanin containing tissues in vitro and in vivo was studied by whole body autoradiography. Manganese was bound to beef eye, human hair and synthetic dopamine melanin, and was taken up in pigmented tissues in mice and a monkey. Long-time exposure to manganese may cause a chronic extrapyramidal disorder. It is suggested that manganese, due to its neurotoxicity, may cause lesions in pigment containing neurones in the substantia nigra secondary to its accumulation on the neuromelanin.

Disposition of some sulphur-containing hexachlorobenzene (HCB) metabolites: affinity of methylsulphonylpentachlorobenzene for the gray matter of brain, the lungs and the adrenal zona reticularis.

The distribution of methylsulphonylpentachloro-[14C]benzene (I), methylthiopentachloro-[14C]benzene (II), 1,4-bis(methylthio)tetrachloro-[14C]benzene (III) and 1,2,4-tris(methylthio)trichloro[14C]benzene (IV) was studied after intravenous injection in mice. The administration of I resulted in a rapid and selective accumulation of radioactivity in the gray matter of the brain, the lung and the zona reticularis of the adrenal cortex. In mice pretreated with metyrapone, or with high dose of unlabelled I, the radioactivity in the tissues was increased, whereas pretreatment with diethyl maleate did not change the disposition of I. In mice injected with II-IV no site of selective accumulation in the tissues was observed. Similarly to I, compounds II-IV were rapidly excreted in bile/faeces and urine. It is proposed that the sulphone group is essential for the high affinity of I for the brain, lung and adrenal cortex.

Accumulation of taurine in the nasal mucosa and the olfactory bulb.

Using whole-body autoradiography of 14C-taurine in mice we have observed a high concentration in the nasal mucosa followed by accumulation in the olfactory bulb at longer survival times. When 14C-taurine was administered in the nasal cavity unilaterally, a high accumulation was observed in the ipsilateral olfactory bulb.

Autoradiography of manganese: accumulation and retention in the pancreas.

By means of whole-body autoradiography, the general distribution of 54MnCl2 was studied in mice and a Marmoset monkey. High accumulation and retention were observed in the pancreas in both species. Gamma counting experiments in mice after a single intravenous injection of 54MnCl2 showed that the level in the pancreas exceeded that of the liver at all survival times (20 min. - 30 days). Also in the monkey, the concentration in the pancreas exceeded that of the liver, and the pancreas had the highest tissue/liver ratio of the organs measured at 24 hours after injection. The high uptake and long retention in the pancreas suggest that manganese is of importance for the pancreatic function but also that the pancreas may be a target organ for manganese toxicity. Positron tomography, using 11C-labelled amino acids, has been found to be a promising diagnostic technique for the study of pancreatic disease. Positron emitting manganese isotopes may be worth further studies as possible agents for pancreatic imaging.

Studies on the melanin affinity of haloperidol.

The general distribution of 3H-haloperidol was studied in mice by means of whole body autoradiography. Extensive accumulation and retention was observed in the melanin containing tissues. In the albino animals uptake was low in the corresponding tissues. Impulse counting of excised tissue pieces showed a maximum concentration in the pigmented eyes 24 hr after a single intravenous injection. In the pigmented eyes, about 1/5 of the radioactivity at 24 hr still remained 90 days after administration. In vitro, haloperidol was found to be bound to beef-eye melanin and dopamine melanin (structurally similar to the melanin present in the pigmented nerve cells in the human substantia nigra). The total binding capacity of beef-eye melanin was approximately twice that of dopamine melanin. Beef-eye melanin bound haloperidol to an extent twice that earlier reported for chlorpromazine and chloroquine, while the binding strengths were of about the same magnitude. Due to its high melanin affinity it is most probable that haloperidol accumulates extensively in the pigmented neurons of the substantia nigra in patients on long term therapy. Other compounds with melanin affinity such as phenothiazine derivatives and manganese are known to produce irreversible extrapyramidal disorders, degeneration and depigmentation of the pigmented nerve cells in the substantia nigra. In conclusion the melanin affinity may be an important factor in the pathogenesis of haloperidol induced tardive dyskinesia.

A whole body autoradiographic study on the distribution of 14C-labelled di-(2-ethylhexyl)phthalate in mice.

Di-(2-ethylhexyl)phthalate (DEHP), a plasticizer used for polyvinylchloride polymers, has been reported to leach from blood transfusion bags and the plastic material in hemodialysis units into the blood. Phthalate esters are known to be hepatotoxic and teratogenic in experimental animals. Reports on the distribution and metabolism of DEHP indicate that the compound in largely excreted from the body within a few days. In the present investigation the distribution and tissue retention after administration of [14C]DEHP (carbonyl-14C or 2-ethylhexyl-1-14c) was studied in pregnant and non-pregnant mice with whole body autoradiography. Initially a high activity was observed in the brown fat, liver, gall bladder, intestinal contents, kidney and urinary bladder. Pretreatment with DEHP, phenobarbital sodium or 3-methylcholanthrene caused a relative increase of the activity in the brown fat, indicating that induced metabolic conversion of DEHP leads to an increased deposition of radioactivity in brown fat. After administration of DEHP (carbonyl-14C), but not DEHP (2-ethylhexyl-1-14C),marked retention was observed in the skin, cartilage and tendons. The mechanism responsible for the slow accumulation in these connective tissues is not known. In the early embryo a high concentration was observed in the neuroepithelium. This pronounced uptake may be correlated to the DEHP-induced malformations exencephaly and spina bifida observed in mice.