Viral co-infection, autoimmunity, and CSF HIV antibody profiles in HIV central nervous system escape.

Antiretroviral therapy (ART) suppresses plasma and cerebrospinal fluid (CSF) HIV replication. Neurosymptomatic (NS) CSF escape is a rare exception in which CNS HIV replication occurs in the setting of neurologic impairment. The origins of NS escape are not fully understood. We performed a case-control study of asymptomatic (AS) escape and NS escape subjects with HIV-negative subjects as controls in which we investigated differential immunoreactivity to self-antigens in the CSF of NS escape by employing neuroanatomic CSF immunostaining and massively multiplexed self-antigen serology (PhIP-Seq). Additionally, we utilized pan-viral serology (VirScan) to deeply profile the CSF anti-viral antibody response and metagenomic next-generation sequencing (mNGS) for pathogen detection. We detected Epstein-Barr virus (EBV) DNA more frequently in the CSF of NS escape subjects than in AS escape subjects. Based on immunostaining and PhIP-Seq, there was evidence for increased immunoreactivity against self-antigens in NS escape CSF. Finally, VirScan revealed several immunodominant epitopes that map to the HIV envelope and gag proteins in the CSF of AS and NS escape subjects. Whether these additional inflammatory markers are byproducts of an HIV-driven process or whether they independently contribute to the neuropathogenesis of NS escape will require further study.

Peroxisome proliferator-activated receptor alpha (PPARalpha) protects against oleate-induced INS-1E beta cell dysfunction by preserving carbohydrate metabolism.

AIMS/HYPOTHESIS: Pancreatic beta cells chronically exposed to fatty acids may lose specific functions and even undergo apoptosis. Generally, lipotoxicity is triggered by saturated fatty acids, whereas unsaturated fatty acids induce lipodysfunction, the latter being characterised by elevated basal insulin release and impaired glucose responses. The peroxisome proliferator-activated receptor alpha (PPARalpha) has been proposed to play a protective role in this process, although the cellular mechanisms involved are unclear. METHODS: We modulated PPARalpha production in INS-1E beta cells and investigated key metabolic pathways and genes responsible for metabolism-secretion coupling during a culture period of 3 days in the presence of 0.4 mmol/l oleate. RESULTS: In INS-1E cells, the secretory dysfunction primarily induced by oleate was aggravated by silencing of PPARalpha. Conversely, PPARalpha upregulation preserved glucose-stimulated insulin secretion, essentially by increasing the response at a stimulatory concentration of glucose (15 mmol/l), a protection we also observed in human islets. The protective effect was associated with restored glucose oxidation rate and upregulation of the anaplerotic enzyme pyruvate carboxylase. PPARalpha overproduction increased both beta-oxidation and fatty acid storage in the form of neutral triacylglycerol, revealing overall induction of lipid metabolism. These observations were substantiated by expression levels of associated genes. CONCLUSIONS/INTERPRETATION: PPARalpha protected INS-1E beta cells from oleate-induced dysfunction, promoting both preservation of glucose metabolic pathways and fatty acid turnover.

Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload.

AIMS/HYPOTHESIS: Saturated fatty acids augment endoplasmic reticulum (ER) stress in pancreatic beta cells and this is implicated in the loss of beta cell mass that accompanies type 2 diabetes. However, the mechanisms underlying the induction of ER stress are unclear. Our aim was to establish whether saturated fatty acids cause defects in ER-to-Golgi protein trafficking, which may thereby contribute to ER stress via protein overload. METHODS: Cells of the mouse insulinoma cell line MIN6 were transfected with temperature-sensitive vesicular stomatitis virus G protein (VSVG) tagged with green fluorescent protein to quantify the rate of ER-to-Golgi protein trafficking. I14 antibody, which detects only correctly folded VSVG, was employed to probe the folding environment of the ER. ER stress markers were monitored by western blotting. RESULTS: Pretreatment with palmitate, but not oleate, significantly reduced the rate of ER-to-Golgi protein trafficking assessed using VSVG. This was not secondary to ER stress, since thapsigargin, which compromises chaperone function by depletion of ER calcium, markedly inhibited VSVG folding and promoted strong ER stress but only slightly reduced protein trafficking. Blockade of ER-to-Golgi protein trafficking with brefeldin A (BFA) was sufficient to trigger ER stress, but neither BFA nor palmitate compromised VSVG folding. CONCLUSIONS/INTERPRETATION: Reductions in ER-to-Golgi protein trafficking potentially contribute to ER stress during lipoapoptosis. In this case ER stress would be triggered by protein overload, rather than a disruption of the protein-folding capacity of the ER.

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets.

AIMS/HYPOTHESIS: Glutamate dehydrogenase (GDH) is a mitochondrial enzyme playing a key role in the control of insulin secretion. However, it is not known whether GDH expression levels in beta cells are rate-limiting for the secretory response to glucose. GDH also controls glutamine and glutamate oxidative metabolism, which is only weak in islets if GDH is not allosterically activated by L-leucine or (+/-)-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH). METHODS: We constructed an adenovirus encoding for GDH to overexpress the enzyme in the beta-cell line INS-1E, as well as in isolated rat and mouse pancreatic islets. The secretory responses to glucose and glutamine were studied in static and perifusion experiments. Amino acid concentrations and metabolic parameters were measured in parallel. RESULTS: GDH overexpression in rat islets did not change insulin release at basal or intermediate glucose (2.8 and 8.3 mmol/l respectively), but potentiated the secretory response at high glucose concentrations (16.7 mmol/l) compared to controls (+35%). Control islets exposed to 5 mmol/l glutamine at basal glucose did not increase insulin release, unless BCH was added with a resulting 2.5-fold response. In islets overexpressing GDH glutamine alone stimulated insulin secretion (2.7-fold), which was potentiated 2.2-fold by adding BCH. The secretory responses evoked by glutamine under these conditions correlated with enhanced cellular metabolism. CONCLUSIONS/INTERPRETATION: GDH could be rate-limiting in glucose-induced insulin secretion, as GDH overexpression enhanced secretory responses. Moreover, GDH overexpression made islets responsive to glutamine, indicating that under physiological conditions this enzyme acts as a gatekeeper to prevent amino acids from being inappropriate efficient secretagogues.

Total spinal anaesthesia in association with insertion of a paravertebral catheter.

An association between intercostal nerve block and the development of a total spinal is rare. Usually, subarachnoid injection is considered to have followed intraneural placement or inadvertent entrance into a dural cuff extending beyond an intervertebral foramen. We report a patient that followed injection of local anaesthetic into a paravertebral catheter sited at surgery in the thoracic paravertebral space of a patient undergoing thoracotomy. This was a life-threatening event that occurred on two occasions before the definitive diagnosis was made. It is considered likely that the paravertebral catheter entered an intervertebral foramen and the tip perforated the dura.

Free radical modulation of insulin release in INS-1 cells exposed to alloxan.

Generation of free radicals is thought to mediate the cytotoxic action of alloxan on the pancreatic beta-cell. In this investigation, the early effects of alloxan on cell function were studied. When INS-1D insulinoma cells were exposed to alloxan (1 mM) for 45 min followed by a 3-hr recovery period, the drug increased basal insulin release while abolishing the effect of glucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was added directly after 5 min of alloxan treatment, whereas KCl-induced secretion was only partially inhibited. In perifusion, alloxan caused transient insulin secretion to 50% of the rates obtained with glucose 30 min later. Under these conditions, epinephrine abolished the stimulation due to both agents. Membrane potential and cytosolic calcium concentrations ([Ca2+]i) were recorded to clarify the action of alloxan. Alloxan-induced insulin release correlated with depolarization of INS-1D cells and a rise in [Ca2+]i. Alloxan did not augment [Ca2+]i in the presence of BHA or the absence of extracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca2+]i, whereas verapamil was ineffective. This suggests that alloxan promotes Ca2+ influx through channels distinct from L-type channels, perhaps through non-selective cation channels. Thus, alloxan causes changes in INS-1D cells prevented by antioxidant treatment, suggesting that free radicals may modulate the ionic permeability leading to functional activation.

Estrogen receptor alpha requires no accessory factors for high-affinity binding to a consensus response element.

Estrogen receptor (ER) alpha is commonly thought to bind to a consensus estrogen response element (ERE) as a homodimer, but previous experiments have not ruled out the presence of other proteins in the ERalpha/ERE complex. To characterize this interaction in more detail, we overexpressed mouse (m) ERalpha in a baculovirus system, using the selective advantage of the apoptosis inhibitor p35. Recombinant mERalpha possesses the predicted molecular weight and binds 17beta-estradiol and an oligonucleotide containing a consensus vitellogenin ERE with high affinity. Over a wide concentration range of mERalpha protein (0.1-50 nM), only one complex was detected between mERalpha and vitellogenin ERE in gel shift assays. The ratio of E2:vitellogenin ERE bound by mERalpha was close to 2:1, and each complex contained only one ERE. The molecular weight of the complex was determined to be 160 000, very close to that predicted for two mERalpha proteins and one ERE oligonucleotide, therefore providing strong evidence that no other proteins were present. Recombinant mERalpha was purified such that it was the only protein observable by silver stain. Purified mERalpha and mERalpha in a nuclear extract behaved identically in Ferguson analysis, providing more evidence that only mERalpha was binding to the ERE. Purified mERalpha bound vitellogenin ERE with high affinity (Kd = 0. 92 +/- 0.20 nM), indicating that no other proteins are necessary for high-affinity mERalpha interaction with a consensus ERE. To determine whether ERalpha in an estrogen-responsive mammalian tissue behaves the same as the overexpressed mERalpha, we tested rat uterine cytosol by Ferguson analysis. ERalpha in rat uterine cytosol behaved identically to overexpressed mERalpha, suggesting that ERalpha in the uterine extract also binds to DNA predominantly as a homodimer with no additional proteins.

Improved insulin secretion of cryopreserved human islets by antioxidant treatment.

The viability of islets of Langerhans prior to grafting is believed to influence the clinical outcome of islet transplantation. To determine whether oxidative stress occurs during the isolation-purification procedure as well as during tissue culture and cryopreservation, we have measured the glutathione redox state (GSH/GSSG) of islets. Human islets were purified by standard techniques from organ donors, cultured, and cryopreserved. Glucose-induced insulin release was monitored in parallel during static incubations to assess the function of the islets. Cultured human islets responded by a 2.2-fold increase in insulin release to a glucose challenge. After cryopreservation the hormonal response was lower. Immediately after islet isolation the GSH/GSSG ratio was 25.2 +/- 5.2, and it increased slightly to 32.0 +/- 6.1 after 1-3 days in tissue culture. The GSH/GSSG decreased significantly after cryopreservation to 12.2 +/- 3.4, suggesting that the freezing and thawing procedures imposed oxidative stress on the islets. To explore this hypothesis further, cryopreserved islets were treated with the antioxidant butylated hydroxyanisole (BHA). Islets exposed to BHA showed an improved glucose-induced insulin release and had an increased insulin content. BHA also protected the islets when they were exposed to alloxan, a free radical generating agent. However, after cryopreservation, BHA treatment did not modify the glutathione redox state. Although the BHA effect could not be explained merely by a change in the glutathione redox state, it is not precluded that redox changes of other cell components ameliorate the glucose sensitivity of the beta cells. Further studies will be needed to determine possible ways of improving islet cryopreservation with antioxidant treatments and particularly, to validate the present observations by in vivo experiments in the context of clinical islet transplantation.

Amyloid in prostatic corpora amylacea.

AIM: To determine the presence and nature of amyloid in prostatic corpora amylacea using immunohistological studies. METHODS: Prostatic tissue from 18 transurethral and two open resection specimens was studied. Paraffin wax embedded tissue sections were stained with haematoxylin and eosin and the alkaline Congo red method with and without previous treatment with potassium permanganate. Sections were also stained with antibodies to amyloid A, beta 2 microglobulin, lambda and kappa light chains, prealbumin IgA, G, M, S100 protein, prostatic specific antigen, amyloid P component and CAM 5.2 (control and blocking studies were performed). RESULTS: The prostatic corpora amylacea universally showed the presence of amyloid. In all instances this contained beta 2 microglobulin. CONCLUSION: Prostatic corpora amylacea represents a localised amyloidosis of beta 2 microglobulin origin that is unrelated to chronic renal failure and haemodialysis.

Systemic amyloidosis of beta 2 microglobulin type.

A patient receiving haemodialysis for 15 years developed systemic amyloidosis of beta 2 microglobulin type. Noticeable deposits of amyloid were present in the myocardium, intervertebral discs, joint cartilages and tendons. Less conspicuous amounts were present in blood vessel walls in the lungs, liver, adrenal glands and brain, and within the stroma of the prostate, testis and kidney, often with foci of calcification.

[Beta 2-microglobulin amyloidosis]. / Amyloidóza beta 2-mikroglobulinového typu.

A 35-year-old man who had been treated by hemodialysis for 15 years suffered from systemic amyloidosis. It was identified as a beta 2-microglobulin type according to immunohistochemistry. Extensive amyloid deposits were found in myocardium, cartilages of intervertebral discs and in carpal tunnel ligament. Less conspicuous deposits comprised blood vessels of lungs, liver, suprarenal glands, brain, and stroma of prostate gland, testicles, kidney, often combined with calcified foci.

Immunohistological characterisation of amyloid deposits in renal biopsy specimens.

The amyloid deposits in 21 renal biopsy specimens were subjected to a detailed immunohistochemical analysis using a panel of antibodies against recognised constituents of tissue amyloid. This was a retrospective study of material originally submitted during the investigation of various renal abnormalities and studied by a routine protocol including histochemistry, electron microscopy, and immunofluorescence. The presence of an amyloid was confirmed in all 21 cases. Seventeen cases contained P component and either amyloid A (AA) (11 cases) or an immunoglobulin light chain associated amyloid (six cases). Four cases contained amyloid material with unusual immunohistochemical findings; one case had AA and P-component (PC) in the interstitium, one case had lambda light chain and beta-2 microglobulin, one case had kappa light chain and Clq, and one case had lambda light chains only. It was possible, therefore, to identify precisely the amyloid constituents and thereby "type" the amyloid by immunohistochemical means. The availability of the antibodies used and their application using these techniques could simplify the confirmation of clinically suspected amyloidosis.

Carpal tunnel syndrome caused by amyloid containing beta 2 microglobulin: a new amyloid and a complication of long term haemodialysis.

Three patients receiving long term haemodialysis treatment for chronic renal failure due to non-amyloid nephropathy developed the carpal tunnel syndrome requiring decompression surgery. The excised material contained amyloid, which by immunocytochemical techniques was shown to contain beta 2 microglobulin. This is, therefore, a new chemical form of amyloid whose deposition is likely to be the cause of osteoarticular and connective tissue disorders, which are being recognised with increasing frequency in patients receiving long term haemodialysis. Raised beta 2 microglobulin levels are known to occur in chronic renal failure, and the molecule is unable to cross conventional dialysis membranes. The importance of beta 2 microglobulin amyloidosis lies in the threat which it poses to the success of long term haemodialysis.