[Contribution of the University Clinical Research Center's laboratoryin the diagnosis of SARS-CoV-2 in Mali]. / Contribution du laboratoire du Centre Universitaire de Recherche Clinique (UCRC) au diagnostic de SRAS-CoV-2 au Mali.

INTRODUCTION: The rapid diagnostic capacities of laboratories in Mali have been an essential element in the response to COVID-19. The University Clinical Research center (UCRC) diagnosed the first cases of Mali COVID-19. OBJECTIVE: The objective was to describe the contribution of the UCRC in the diagnosis of Covid-19 and to clinically and epidemiologically characterize the patients tested in the UCRC laboratory. MATERIALS AND METHODS: A cross-sectional study was conducted during eight months of intense activity. The samples were sent from the National Institute of Public Health (INSP) to the UCRC. RESULTS: The UCRC tested 12,406 contacts and suspected samples and confirmed the diagnosis in 1091 patients, or 9%. The most common symptoms were cough (48.78%), headache (34.14%), fatigue / weakness (34.14%), while (33.33%) of the patients were asymptomatic. The sample positivity rate among new cases decreased from May to September 2020, despite almost 230% of the number of samples tested. CONCLUSION: The laboratory played a major role in the response and there may be a low transmission of the virus in the Malian community.

INTRODUCTION: Les capacités de diagnostic rapide des laboratoires au Mali ont été un élément essentiel dans la riposte contre la COVID-19. Le Centre Universitaire de Recherche Clinique (UCRC)a diagnostiqué les premiers cas du Mali. OBJECTIF: Etait de décrire l'apport de l'UCRC dans le diagnostic de la Covid-19 et de caractériser cliniquement et épidémiologiquement les patients testés au laboratoire de l'UCRC. MATÉRIELS ET MÉTHODES: Une étude transversale a été conduite pendant huit mois d'activité intense. Les échantillons ont été envoyés de l'Institut National de Santé Publique (INSP) à l'UCRC. RÉSULTATS: L'UCRC a testé 12 406 échantillons contacts et suspects et a confirmé le diagnostic chez 1091 patients soit 9%. Les symptômes les plus rencontrés ont été la toux (48,78%), les maux de tête (34,14%), la fatigue/faiblesse (34,14%), tandis que (33,33%) des patients étaient asymptomatiques. Le taux de positivité des échantillons a diminué entre mai et août et avec une légère diminution en septembre 2020,avec près de 230% du nombre d'échantillons testés. CONCLUSION: Le laboratoire a joué un grand rôle dans la riposte et il y'aurait une faible transmission du virus dans la communauté Malienne.

Second-line antiretroviral therapy failure and characterization of HIV-1 drug resistance patterns in children in Mali.

INTRODUCTION: In recent years, children born to HIV-infected mothers have been receiving antiretroviral treatment (ART) with limited or no virologic monitoring, which increases the likelihood of development and accumulation of drug resistance mutations, which itself may limit the effectiveness of future ART. The objective of this study was to evaluate the prevalence of resistance mutations in children infected with HIV-1 experiencing virological failure to second-line ART in the Pediatric Department of Gabriel Touré Hospital in Mali. METHODS: Children aged from 5 to 18 infected with HIV-1 on second-line antiretroviral therapy and whose viral load was greater than 1000 copies/mL after observance reinforcement were enrolled. The protease and reverse transcriptase genes were sequenced with ViroSeq®. The results were interpreted according to the last version of the Stanford algorithm in 2018. The study was approved by the Ethics Committee of the Faculty of Medicine and Dentistry, University of Sciences, Techniques and Technologies of Bamako (Mali). RESULTS: Of 216 children, 33 (15.3%) who had a viral load (VL)>1000 copies/mL in second line were recruited and included in the study. The median plasma viral load was 77,000 copies/mL [IQR (28,000-290,000)] and the median CD4 cell count was 310 cells/mm3 [IQR (152-412)]. The median age was 12 years; 48.5% of patients were treated with a combination of stavudine/lamivudine/nevirapine (Triomune®) for first-line treatment and 60.6% with abacavir/lamivudine/lopinavir/ritonavir for the second-line ART. The median treatment duration was 8.5 years [range, 3-13]. Of the 33 children whose treatment failed, the predominant HIV-1 subtype was CRF02_AG (66.7%). The prevalence of resistance to ART classes was 60.61% (20/33) to nucleoside reverse transcriptase inhibitors (NRTIs), 54.51% (18/33) to nonnucleoside reverse transcriptase inhibitors (NNRTIs), and 51.52% (17/33) to protease inhibitors (PIs). Of the patients studied, 90.9% were exposed to lopinavir/ritonavir (LPV/r) but only 15.2% (5/33) developed resistance to LPV/r. CONCLUSIONS: This study demonstrated that LPV/r remains active in most patients after second-line ART failure. In children whose second-line ART fails, particular attention should be paid to their ART and adherence history when considering the next treatment option.

Ultrafast photoinduced melting of a spin-Peierls phase in an organic charge-transfer compound, K-tetracyanoquinodimethane.

Ultrafast photoinduced phase transition in a spin-Peierls (SP) system of K-tetracyanoquinodimethane (K-TCNQ) was studied by femtosecond (fs) reflection spectroscopy. Photocarriers destabilize the SP phase, resulting in a decrease in molecular dimerization within 400 fs. Such a melting of the SP phase drives three kinds of coherent oscillations. By comparing the oscillations with the Raman bands activated by the dimerization, we show that the oscillation of 20 cm-1 is due to an LO phonon, and it plays an important role for the stabilization of the SP phase.

A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes.

We have isolated a novel gene, hKCa4, encoding an intermediate conductance, calcium-activated potassium channel from a human lymph node library. The translated protein comprises 427 amino acids, has six transmembrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shares 41-42% similarity at the amino acid level with three small conductance calcium-activated potassium channels cloned from brain. Northern blot analysis of primary human T lymphocytes reveals a 2.2-kilobase transcript that is highly up-regulated in activated compared with resting cells, concomitant with an increase in KCa current. hKCa4 transcript is also detected by Northern blots or by polymerase chain reaction in placenta, prostate, thymus, spleen, colon, and many cell lines of hematopoietic origin. Patch-clamp recordings of hKCa4-transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassium current that is blocked by externally applied tetraethylammonium (Kd = 30 +/- 7 mM), charybdotoxin (Kd = 10 +/- 1 nM), and clotrimazole (Kd = 387 +/- 34 nM), but is resistant to apamin, iberiotoxin, kaliotoxin, scyllatoxin (Kd > 1 microM), and margatoxin (Kd > 100 nM). Single hKCa4 channels have a conductance of 33 +/- 2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (Kd = 270 +/- 8 nM) with a highly cooperative interaction of approximately three calcium ions per channel. These properties of the cloned channel are very similar to those reported for the native KCa channel in activated human T lymphocytes, indicating that hKCa4 encodes this channel type.

Extracellular site for econazole-mediated block of Ca2+ release-activated Ca2+ current (Icrac) in T lymphocytes.

1. Standard whole cell patch clamp recording techniques were used to study the pharmacological characteristics and site of econazole-mediated inhibition of calcium release-activated calcium current (Icrac) in the human leukaemic T cell line, Jurkat. 2. Extracellularly applied econazole blocked Icrac in a concentration-dependent manner (IC50 approximately 14 microM). Block developed over a relatively slow timecourse of 30-60 s (10 microM), and only partially reversed over minutes. 3. Econazole dialysed from the pipette into the cytosol at concentrations ranging from 0.1 to 30 microM did not reduce Icrac, or quantitatively affect Icrac block by extracellularly applied econazole. 4. A less lipophilic quaternary iodide derivative of econazole was synthesized to retard absorption through the cell membrane. When applied extracellularly, this compound blocked Icrac in a concentration-dependent manner with onset kinetics comparable to econazole. 5. Results with intracellularly dialysed econazole and the quaternary econazole derivative provide convergent evidence that econazole blocks Icrac via an extracellular interaction. 6. The inability of intracellularly applied econazole to inhibit Icrac argues against the notion that econazole inhibits capacitative Ca2+ entry pathways secondary to its known inhibitory effects on cytochrome P-450.

Calcium-dependent enhancement of depletion-activated calcium current in Jurkat T lymphocytes.

We have obtained evidence that the Ca(2+)-selective current activated by Ca2+ store depletion (Ca2+ release-activated Ca2+ current; Icrac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca2+ itself. Whole cell patch clamp experiments employed high cytosolic Ca(2+)-buffering conditions to passively deplete Ca2+ stores. Rapidly switching to nominally Ca(2+)-free extracellular buffer instantaneously reduced Icrac measured at -100 mV to leak current level. Unexpectedly, readmission of 2 mM Ca2+ instantaneously restored only 38 +/- 5% (mean +/- SEM, n = 9) of the full Icrac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca2+ chelators did not alter this process, and inorganic Icrac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca2+: introduction of the permeant ions, Ba2+ or Sr2+, failed to invoke time-dependent Icrac reappearance. Moreover, equimolar substitution of Ba2+ for Ca2+ initially produced Ba2+ current of similar magnitude to the full Ca2+ current, but the Ba2+ current decayed monotonically to < 50% of its initial amplitude in < 20 sec. Conversely, return to Ca2+ produced a time-dependent increase in Icrac to its larger Ca2+ permeation level. Thus Ca2+ appears to selectively promote a reversible transition of Icrac that results in larger current flux, and at least partially explains the selectivity of this current for Ca2+ over other divalent ions.

Excitable properties and underlying Na+ and K+ currents in neurons from the guinea-pig jugular ganglion.

Neurons in the superior vagal (jugular) ganglion relay afferent information from thoracic visceral organs and may be important in inflammatory processes due to the peripheral release of bioactive neuropeptides such as substance P. We characterized the excitable properties and underlying voltage-gated Na+ (INa) and K+ (IKv) currents in acutely dissociated guinea pig jugular ganglion neurons with microelectrode and whole-cell patch-clamp recording techniques. Current clamp recordings revealed a resting potential of approx. -55 mV and input resistance of approx. 100 M ohms. Brief depolarizing steps evoked an overshooting action potential (approx. 2 ms duration), fast (< 20 ms duration) afterhyperpolarization (AHPF) sequence in all neurons, followed by a slow (> 1 s) Cd(2+)-sensitive afterhyperpolarization (AHPS) in 45% of the neurons. The AHPS was implicated in limiting repetitive action potential firing during maintained depolarizing steps. The action potential in 15/17 neurons, and a major component of the whole cell INa in 13/13 neurons were insensitive to TTX (1-10 microM), indicating that jugular neurons express predominantly a TTX-resistant type of INa. Cd2+ (200 microM) did not affect action potential repolarization, while tetraethylammonium (TEA; 10 mM) in the presence of Cd2+ markedly prolonged action potential repolarization, and blocked the AHPF in 11/11 neurons. This suggested that the action potential repolarization and the AHPF are mediated by IKv, with little contribution by Ca(2+)-dependent IK (IK(Ca)). Whole cell IKv activated rapidly (tau < 1.5 ms), and inactivated variably over a time period of seconds. IKv activation and inactivation voltage dependencies and TEA sensitivity were compatible with its availability during the action potential and AHPF. Only 1/26 neurons exhibited current with the rapid inactivation kinetics and voltage-dependencies characteristic of classic IA-type current. These results highlight differences in the properties of jugular neurons (e.g., deficiency of rapid IA, and lack of a TTX-sensitive subpopulation), relative to those known for other visceral and somatic afferents, and thus provide a basis for further functional studies.

Calcium current characterization in dissociated adult guinea-pig jugular ganglion neurons.

Voltage-dependent calcium (Ca2+) channel currents in freshly dissociated adult guinea-pig jugular ganglion neurons were examined and characterized using the whole-cell patch-clamp technique. Electrophysiological analysis demonstrated a high-threshold current, but no low-threshold or T-type current. A fraction of the total Ca2+ current was inhibited by omega (omega)-conotoxin GVIA (Cg (inTX; 10 microM); the dihydropyridine antagonist nifedipine (NIF; 10 microM), inhibited a large fraction of the CgTX-insensitive current. The remaining CgTX/NIF-insensitive current was completely inhibited by omega-agatoxin IVA (AgIVA; 100 nM). These results demonstrated that the whole-cell Ca2+ channel current consisted only of N-, L- P-type components. Of these currents, only the L-type was partially inhibited by both histamine and carbachol (0.01-100 microM).

Guinea pig visceral C-fiber neurons are diverse with respect to the K+ currents involved in action-potential repolarization.

1. Intracellular recordings were made from C-fiber neurons identified by antidromic conduction velocity in intact guinea pig nodose ganglia maintained in vitro, and whole-cell patch clamp recordings were made from dissociated guinea pig nodose neurons to investigate the contribution of various K+ conductances to action-potential repolarization. 2. The repolarizing phase of the intracellularly recorded action potential was prolonged in a concentration-dependent manner by charybdotoxin (Chtx; EC50 = 39 nM) or iberiatoxin (Ibtx; EC50 = 48 nM) in a subpopulation of 16/36 C-fiber neurons. In a subset of these experiments, removal of extracellular Ca2+ reversibly prolonged action-potential duration (APD) in the same 4/9 intracellularly recorded C-fiber neurons affected by Chtx (> or = 100 nM). These convergent results support that a Ca(2+)-activated K+ current (IC) contributes to action-potential repolarization in a restricted subpopulation of C-fiber neurons. 3. Tetraethylammonium (TEA; 1-10 mM) increased APD considerably further in the presence of 100-250 nM Chtx or Ibtx, or in nominally Ca(2+)-free superfusate in 14/14 intracellularly recorded C-fiber neurons. TEA affected APD similarly in subpopulations of neurons with and without IC, suggesting that a voltage-dependent K+ current (IK) contributes significantly to action-potential repolarization in most nodose C-fiber neurons. 4. Substitution of Mn2+ for Ca2+ reduced outward whole-cell currents elicited by voltage command steps positive to -30 mV (2-25 ms) in a subpopulation of 21/36 dissociated nodose neurons, supporting the heterogeneous expression of IC. The kinetics of outward tail current relaxations (tau s of 1.5-2 ms) measured at the return of 2-3 ms depolarizing steps to -40 mV were indistinguishable in neurons with and without IC, precluding a separation of the nodose IC and IK by a difference in deactivation rates. 5. Chtx (10-250 nM) reduced in a subpopulation of 3/8 C-fiber neurons the total outward current elicited by voltage steps depolarized to -30 mV in single microelectrode voltage-clamp recordings. TEA (5-10 mM) further reduced outward current in the presence of 100-250 nM Chtx in all eight experiments. The Chtx-sensitive current was taken to represent IC, and the TEA-sensitive current, the IK component contributing to action-potential repolarization. 6. Rapidly inactivating current (IA) was implicated in action-potential repolarization in a subpopulation of intracellularly recorded C-fiber neurons. In 4/7 neurons, incremented hyperpolarizing prepulses negative to -50 mV progressively shortened APD.(ABSTRACT TRUNCATED AT 400 WORDS)

A retrograde labeling technique for the functional study of airway-specific visceral afferent neurons.

The development of a method is described whereby primary afferent neurons that specifically innervate the airways in the guinea pig can be retrogradely labeled, acutely dissociated and studied functionally with electrophysiological techniques. Following administration of either dextran-tetramethylrhodamine, Fast Blue, or Fluorogold dye into the tracheal lumen, dye-labeled neurons can be visualized in 100 microns serial nodose ganglion sections. Control experiments show that labeling does not result from the undesirable spread of the dyes to target innervation fields in the gastrointestinal (GI) or cardiovascular (CV) systems. Neuronal somata retain dye label when acutely dissociated. Microelectrode studies provide evidence that the presence of the Rhodamine dye label and its fluorescent excitation neither alter basic electrophysiological membrane parameters nor the chemoreceptive properties of isolated neurons. Thus this new method will allow the isolation of individual airway-specific primary visceral afferent neurons for functional studies with multidisciplinary techniques.

Probing the bradykinin receptor: mapping the geometric topography using ethers of hydroxyproline in novel peptides.

Five decapeptides were prepared, each having the generic primary sequence D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-X7 -Y8-Arg9. A C-terminal beta-turn was anticipated when X was an alkyl ether of D-4-hydroxyproline in either the cis or trans geometric state and Y was either a Tic or Oic residue. Whereas cis ethers have only very weak receptor affinities, the trans ethers are significantly more potent in binding to guinea pig smooth muscle having Ki values as low as 0.16 nM. Notably, these peptides do not contain a D-aromatic amino acid at position 7 of the primary sequence.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin, a potent antagonist of smooth muscle BK2 receptors and BK3 receptors.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin (NPC 16731) inhibited bradykinin (BK) binding and BK-induced contraction in guinea-pig ileum, being markedly more potent than D-Phe7-BK analogues as a BK2 receptor antagonist. In isolated trachea NPC 16731, unlike other BK2 antagonists, inhibited BK binding and BK-induced contraction, and 45Ca2+ efflux in tracheal smooth muscle cells. That NPC 16731 potently inhibits BK effects in trachea provides further evidence for the existence of the airway BK3 receptor.

Effects of epithelium removal on relaxation of airway smooth muscle induced by vasoactive intestinal peptide and electrical field stimulation.

1. We have studied the effect of epithelium removal on relaxation of guinea-pig isolated tracheal smooth muscle induced by vasoactive intestinal peptide (VIP) or stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves. Also examined were the effects of inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE). 2. Epithelium removal produced a 3.6 +/- 0.4 fold leftward shift in the VIP concentration-response curve. The supersensitivity to VIP, following epithelium removal was abolished by phosphoramidon or thiorphan (NEP inhibitors), but unaffected by captopril (an ACE inhibitor). In intact trachea, the NEP inhibitors produced leftward shifts in the VIP curves similar to those produced by epithelium removal. 3. In contrast to responses to exogenous VIP, neurogenic NANC inhibitory responses to electrical field stimulation were affected neither by epithelial denudation nor by the peptidase inhibitors. 4. As in previous studies, epithelium removal increased tracheal sensitivity to isoprenaline. This was not altered by pretreatment with a cocktail of peptidase inhibitors. Thus, the effect of the NEP inhibitors on responses to VIP appears to be relatively specific. 5. These data indicate that exogenous VIP is a substrate for airway NEP, since inhibition of the enzyme potentiates the peptide. This is further evidence that the airway epithelium provides a source for the metabolism of mediators. 6. In guinea-pig trachea the NEP responsible for cleaving VIP may be located largely in the epithelial layer, since NEP inhibition was without effect on sensitivity to VIP in epithelium-denuded preparations. If VIP is a NANC inhibitory neurotransmitter in this tissue its degradation endogenously does not appear to involve epithelial NEP.

[Arg1-D-Phe7]-substituted bradykinin analogs inhibit bradykinin- and vasopressin-induced contractions of uterine smooth muscle.

The purpose of the present study was to examine the tissue selectivity of several [Arg1-D-Phe7]-substituted analogs of bradykinin. Unlike D-Arg-[Hyp3-D-Phe7]-bradykinin (NPC567), which antagonizes bradykinin-induced contractions both in rat isolated uterus and guinea pig ileum, [D-Nal1-Thi5,8-D-Phe7]-bradykinin (NPC573) was active only in uterine smooth muscle. Binding studies revealed that, unlike several [D-Phe7]-substituted analogs, including NPC567, NPC573 competed with radiolabeled bradykinin neither at receptors in uterus nor ileum. Moreover, no [Arg1-D-Phe7]-substituted analog competed with bradykinin binding in guinea pig ileum, suggesting that these agents, which inhibit uterine but not ileal contractions to bradykinin, may not be bradykinin receptor antagonists. NPC573 inhibited [Arg8]-vasopressin-induced contraction of the uterus more potently than it did bradykinin, although NPC573 (and other [Arg1-D-Phe7]-substituted analogs tested) did not inhibit binding of a vasopressin antagonist either in uterus or liver membranes. We therefore suggest that [Arg1-D-Phe7]-substituted analogs of bradykinin inhibit contractions of uterine smooth muscle by a mechanism other than receptor antagonism. In addition, the tissue selectivity of these agents suggests that the mechanisms underlying bradykinin's contractile effect in uterus are different than in intestinal smooth muscle.

D-Arg-[Hyp3-D-Phe7]-bradykinin, a bradykinin antagonist, reduces mortality in a rat model of endotoxic shock.

The kallikrein-kinin system is activated during endotoxic shock, suggesting that bradykinin plays a role in the pathology of this disease. To test this hypothesis, a bradykinin antagonist, D-Arg-Hyp3-D-Phe7-bradykinin (NPC 567), was studied in conscious, chronically catheterized rats undergoing lipopolysaccharide (LPS)-induced endotoxic shock. LPS treatment resulted in an increase in circulating bradykinin from less than 23 pg/ml to 144 +/- 18 pg/ml at 1 hr. Intravenous administration of LPS resulted in a 38% drop in mean arterial pressure at 1 hr which was partially reversed by NPC 567. NPC 567 did not affect the moderate tachycardia observed following LPS. NPC 567 infusion at 8 nmol/kg/min dramatically reduced mortality from 100% to 50% at 24 hr (P less than 0.01). In response to LPS, blood thromboxane B2 (TXB2) rose from less than 200 pg/ml to 2,298 +/- 64 pg/ml, while 6-keto-prostaglandin-F1 alpha (6kPGF1 alpha) rose from 289 +/- 23 pg/ml to 7,927 +/- 822 pg/ml. NPC 567 reduced the rise in 6kPGF1 alpha by 42% (P less than 0.05), without affecting TXB2. In summary, NPC 567 reduced mortality in rats treated with LPS, reduced the rise in 6kPGF1 alpha and partially reversed the hypotensive effects. These results suggest that bradykinin plays a significant role in the pathology of endotoxic shock.

D-Phe7-substituted peptide bradykinin antagonists are not substrates for kininase II.

The bradykinin receptor antagonists [D-Phe7]bradykinin, D-Arg[Hyp3,D-Phe7]bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]bradykinin were tested for their ability to serve as substrates for kininase II (angiotensin converting enzyme) purified from rabbit lung. By HPLC, the peptides were not measurably degraded over 30 minutes. Under identical conditions, bradykinin was completely degraded to bradykinin (1-7). When hippuryl-His-Leu was used as a substrate for kininase II, the D-Phe7-substituted bradykinins acted as weak noncompetitive inhibitors. While the peptides were poor substrates for kininase II, they were short-lived when injected intravenously. D-Arg[Hyp3,D-Phe7]bradykinin was completely degraded to small fragments in less than 2 minutes. In diluted serum in vitro, a single product was observed with elution consistent with loss of arginine, suggestive of metabolism by kininase I.

Supplemental zinc restores antibody formation in cultures of aged spleen cells. III. Impairment of II-2-mediated responses.

The effects of zinc on interleukin-2(IL-2)-dependent T cell responses of lymphocytes from immunodepressed aged mice and from young adult animals were studied. Concentrations of zinc which have been shown to restore antibody formation in cells from aged mice and to increase the production of Il-1 and Il-4 inhibited the production of Il-2. Cells from both young adults and aged mice were inhibited similarly. Zinc also impaired the ability of aged T cells to proliferate in response to concanavalin A and exogenous Il-2, but enhanced the proliferation of similarly activated splenic cultures containing both T and B cells. Cultures of isolated B lymphocytes produced antibody to sheep red blood cells if the cells were provided with supplemental zinc and Il-1. In contrast, recombinant Il-2 with or without zinc did not activate antibody formation. The results support the premise that the restorative effects of zinc are independent of Il-2 and that Il-2 is not a necessary mediator for antibody production in the aged.