Inverse relationship between erythrocyte size and platelet reactivity in elderly.

Previous work indicates that erythrocytes (RBCs) accumulate ß-amyloid X-40 (Aß40) in individuals with Alzheimer disease (AD) and to a lesser extent in healthy elderly. The toxin damages RBCs and increases their mean corpuscular volume (MCV). Furthermore, AD platelets demonstrate lower reactivity. This study investigated interactions between RBCs and platelets. Older individuals with moderate hypertension (n = 57) were classified into two groups, depending on MCV in whole blood: The MCVhigh group comprised individuals with higher MCV (n = 27; 97 ± 3(SD) fl) and MCVlow group had relatively lower MCV (n = 30; 90 ± 3(SD) fl). Flow cytometry was used to determine platelet reactivity, i.e., the surface binding of fibrinogen after provocation. Adenosine diphosphate (ADP) and a thrombin receptor-activating protein (TRAP-6) were used as agonists. Subsequently, blood cells were divided according to density into 17 subfractions. Intra-RBC Aß40 content was analyzed and in all platelet populations surface-bound fibrinogen was determined to estimate platelet in vivo activity. We found Aß40 inside RBCs of approximately 50% of participants, but the toxin did not affect MCV and platelet reactivity. In contrast, MCV associated inversely with platelet reactivity as judged from surface-attached fibrinogen after ADP (1.7 µmol/L) (p < 0.05) and TRAP-6 provocation (57 µmol/L (p = 0.01) and 74 µmol/L (p < 0.05)). In several density fractions (nos. 3, 4, 8, 11-13 (p < 0.05) and nos. 5-7 (p < 0.01)) MCV linked inversely with platelet-attached fibrinogen. In our community-dwelling sample, enhanced MCV associated with decreased platelet reactivity and lower in vivo platelet activity. It resembles RBCs and platelet behavior in AD-type dementia.

Underlying theory of a model for the Renner-Teller effect in tetra-atomic molecules: X(2)Πu electronic state of C2H2(+).

In the present study, we prove the plausibility of a simple model for the Renner-Teller effect in tetra-atomic molecules with linear equilibrium geometry by ab initio calculations of the electronic energy surfaces and non-adiabatic matrix elements for the X(2)Πu state of C2H2 (+). This phenomenon is considered as a combination of the usual Renner-Teller effect, appearing in triatomic species, and a kind of the Jahn-Teller effect, similar to the original one arising in highly symmetric molecules. Only four parameters (plus the spin-orbit constant, if the spin effects are taken into account), which can be extracted from ab initio calculations carried out at five appropriate (planar) molecular geometries, are sufficient for building up the Hamiltonian matrix whose diagonalization results in the complete low-energy (bending) vibronic spectrum. The main result of the present study is the proof that the diabatization scheme, hidden beneath the apparent simplicity of the model, can safely be carried out, at small-amplitude bending vibrations, without cumbersome computation of non-adiabatic matrix elements at large number of molecular geometries.

The impact of ethical implications intertwined with tuberculosis household contact investigation: a qualitative study.

Background: Household contact investigation (HCI) is an effective and widely used approach to identify persons with tuberculosis (TB) disease and infection, globally. Despite widespread recommendations for the use of HCI, there remains poor understanding of the impact on and value of contact investigation for participants. Further, how HCI as a practice impacts psychosocial factors, including stigma and possible unintended disclosure of illness among persons with TB, their families, and communities, is largely unknown. Methods: This exploratory qualitative study nested within a randomized trial (ClinicalTrials.gov: NCT04520113, 17 August 2020) was conducted in South Africa to understand the impacts of HCI on index patients living with TB and their household contact persons in two rural districts in the Limpopo province (Vhembe and Capricorn) and Soshanguve, a peri-urban township in Gauteng province. People with TB and household members of people with TB were recruited to participate in in-depth interviews and focus group discussions using semi-structured guides. We explored individual, interpersonal, and community-level perceptions of potential impacts of household contact investigation to elucidate their perceptions of HCI. Thematic analysis identified key themes. Results: Twenty-four individual interviews and six focus group discussions (n=39 participants) were conducted. Participants viewed HCI as an effective approach to finding TB cases, helpful in educating households about TB symptoms and reducing barriers to health-related services. At the interpersonal level, HCI aided people with TB in safely disclosing their TB status to family members and facilitated family and social support for accountability. The introduction of HIV testing during HCI was reported by some participants as making household members slightly uncomfortable, decreasing interest in household members being tested for TB. HCI negatively impacted community-level TB and HIV-related stigma due to healthcare worker visibility at home. Conclusion: Our data suggests varying impacts of HCI on people with TB, their families and interpersonal relationships, and communities, highlighting the importance of considering approaches that address concerns about community stigma and HIV testing to enhance acceptance of HCI.

Platelets and acute cerebral infarction.

Stroke is worldwide a leading cause of death and disability. Its etiology is regarded as heterogeneous. Platelets are implicated in its pathophysiology, but our understanding of their specific role is incomplete. Only sparse and conflicting information exists about platelet reactivity and activity in acute stroke. Some scientists take the view that platelets activate in conjunction with acute cerebral infarctions. Others put forward evidence corroborating the contrary notion. Increased soluble P-selectin as a sign of platelet and/or endothelial activity seems to be a feature of the disease. The latter point of view is opposed by other researchers. Due to these conflicting opinions, this study is devoted to platelet characteristics in acute cerebral infarctions. We studied subjects (n = 72; age 74 ± 10(SD) years; 31 females) having acute stroke. As controls served atrial fibrillation (AF) patients (n = 58; age 69 ± 7(SD) years; 12 females) subject to electrical cardioversion, a flow cytometer was put to use for measuring platelet reactivity and activity. After agonist provocation, both platelet bound P-selectin and fibrinogen were employed as estimates of platelet reactivity. Dilutions of a thrombin-receptor-activating peptide (TRAP-6) (74 and 57 µmol/l) (P-selectin and fibrinogen) and ADP (8.5 and 1.7 µmol/l) (fibrinogen only) were put to use as platelet agonists. Membrane-bound P-selectin without agonist stimulation served as a measure of in vivo platelet activation. Soluble P-selectin, as determined from a commercial ELISA, was used to assess platelet and/or endothelial activity. In acute stroke neither platelet-bound P-selectin nor fibrinogen after stimulation, i.e. reactivity, differed from AF controls. In contrast, lower platelet activity as judged from surface attached and circulating P-selectin without agonist stimulation proved to be a feature of cerebral infarctions. The p-values were p < 0.001 and p < 0.01, respectively. It is concluded that acute stroke is not associated with platelet reactivity platelets circulate less activated during the disease. It is evident that the mechanisms reflecting platelet reactivity and activity being investigated in this study play minor roles in stroke pathophysiology. New powerful platelet inhibitory drugs are currently introduced. To avoid major bleeding studies on platelet, behavior in acute stroke are necessary before including these medications in stroke treatment protocols.

Cytotoxic effects of palladium (II) and platinum (II) complexes with O,O'-dialkyl esters of (S,S)-ethylenediamine-N,N'-di-2-(4-methyl) pentanoic acid on human colon cancer cell lines.

PURPOSE: As novel therapeutic agents relevant to colon cancer therapy are explored continuously, we tested 4 R2edda-type ligand precursors O,O'-dialkyl esters of (S,S)-ethylenediamine-N,N'-di-2-(4-methyl)pentanoic acid (L1.2HCl-L4.2HCl) and corresponding palladium(II) and platinum(II) complexes against the human colon cancer cell lines CaCo-2, SW480 and HCT116. METHODS: The effects of the tested compounds on cell viability were determined using MTT colorimetric technique. RESULTS: Analysis of cancer cell viability showed that all tested ligand precursors, palladium(II) and platinum(II) complexes were cytotoxic on human colon cancer cells in dose-dependent manner. The cytotoxic activity of all palladium(II) and platinum(II) complexes toward selected cancer cells was significantly higher in comparison to cisplatin. Among the tested platinum(II) and palladium(II) complexes the lowest activity was observed for the compounds with the shortest ester chain and the highest activity was noted for palladium(II) complex No.2 with the n-Pr group in ester chain and for platinum(II) complex No.7 with the n-Bu group in ester chain. CONCLUSION: Palladium(II) complex No.2 and platinum(II) complex No.7 seem to be good candidates for future pharmacological evaluation in the field of colon cancer research and treatment.

Low-density platelet populations demonstrate low in vivo activity in sporadic Alzheimer disease.

Platelets contain a substantial quantity of amyloid-precursor protein (APP) and ß-amyloid. However, despite the large importance of APP and ß-amyloid to dementia, little is known about platelets in sporadic Alzheimer dementia (AD). Furthermore, platelet heterogeneity influences human pathology and has been described to affect the progression of AD. This study investigated AD platelets with respect to density diversity and in vivo activity associated with density sub-fractions. We included 39 AD patients and used, as controls, 22 elderly individuals without apparent memory disorder. A continuous Percoll™ gradient covering the density span 1.04-1.09 kg/l provided the basis to divide platelets of whole blood into density fractions (n = 16). All platelet populations were evaluated accordingly. Platelet counts were determined electronically. A flow-cytometer was put to use to measure surface-bound fibrinogen as a measure of platelet in vivo activity. Samples obtained from patients diagnosed with sporadic AD contained platelets (fractions numbers 4-16) that circulated with significantly less surface-bound fibrinogen, i.e., their platelet activation in vivo was reduced, compared with controls. In particular, highly significant differences (p < 0.001) were obtained for the six less dense platelet populations (fractions numbers 11-16) when comparing sporadic AD with controls. In contrast, the densest AD platelets in fractions numbers 1-3 did not differ significantly from control cells with respect to in vivo platelet-bound fibrinogen. It is concluded that sporadic AD is characterized by lower density platelet populations that, while circulating, exhibited reduced activation. The clinical significance of this finding is unclear but these results suggest the importance of platelet heterogeneity in dementia as a topic for further investigation.

A new semiquantitative method for evaluation of metastasis progression.

PURPOSE: Although recent technical advancements are directed toward developing novel assays and methods for detection of micro and macro metastasis, there are still no reports of reliable, simple to use imaging software that could be used for the detection and quantification of metastasis in tissue sections. We herein report a new semiquantitative method for evaluation of metastasis progression in a well established 4T1 orthotopic mouse model of breast cancer metastasis. METHODS: The new semiquantitative method presented here was implemented by using the Autodesk AutoCAD 2012 program, a computer-aided design program used primarily for preparing technical drawings in 2 dimensions. RESULTS: By using the Autodesk AutoCAD 2012 software- aided graphical evaluation we managed to detect each metastatic lesion and we precisely calculated the average percentage of lung and liver tissue parenchyma with metastasis in 4T1 tumor-bearing mice. The data were highly specific and relevant to descriptive histological analysis, confirming reliability and accuracy of the AutoCAD 2012 software as new method for quantification of metastatic lesions. CONCLUSION: The new semiquantitative method using AutoCAD 2012 software provides a novel approach for the estimation of metastatic progression in histological tissue sections.

Impact of Dermatophagoides pteronyssinus mite body raw material on house dust mite allergy diagnosis in a Serbian population.

House dust mite (HDM) allergy has different clinical and immunological patterns in different geographic regions. The impact of raw material of commercial Dermatophadoides pteronyssinus (Acari: Pyroglyphidae) mite bodies on the quality of allergen extracts for allergy diagnosis in the Serbian population has not been previously evaluated. House dust mite bodies obtained from manufacturers in Europe, South America and Australia were used in the preparation of allergen extracts for in vivo diagnosis and serological analysis in a group of 14 HDM-allergic adults. In the group of mite-allergic patients, there was no statistically significant difference in skin test reactivity (Wilcoxon matched pairs test) among the three HDM body extract preparations. In a CAP inhibition assay, two extracts (A and C) achieved maximum inhibition of >90%, whereas extract B demonstrated a different inhibition slope and lower inhibition potential (80%). However, a remarkable difference in immunoglobulin E reactivity using Western blot analysis with individual patients' sera was observed in one of the preparations (extract B). These findings emphasize the need for the careful selection of starting material for the preparation of HDM diagnostic reagents intended for use in patients from geographically distinct regions as these preparations can have implications on the selection criteria for patient-tailored immunotherapy of HDM allergy.

Interleukin-17 may be a valuable serum tumor marker in patients with colorectal carcinoma.

The promotion of tumor growth is due to a combination of several mechanisms, including angiogenesis and the abundance of cell-derived inflammatory cytokines. The aim of this study was to investigate the serum levels of interleukin 17 (IL-17) and the expression of p53 and Vascular Endothelial Growth Factor (VEGF), in order to determine the relationship between these markers and serum IL-17 levels in patients with colorectal carcinoma. Serum levels of the proinflammatory cytokine IL-17 in patients with colorectal carcinoma (CRC) (n=40) and in a healthy group (n=37) were analysed by ELISA. Surgically resected specimens of 59 colorectal carcinomas were studied by immunohistochemical staining for VEGF and p53. Analyses by ELISA showed significantly higher IL-17 serum levels in patients with colorectal carcinoma than in control subjects (IL-17; mean 128.52+/-47.62 pg/ml vs. mean 101.91+/-22.46 pg/ml; p=0.022). We also found an inverse correlation between p53 expression and the level of IL-17 in the serum of patients with CRC. In fact, the serum concentration of IL-17 was significantly higher in patients who did not express p53 (p=0.023). There was no significant correlation between the expression of p53 and VEGF. However, concomitant expression of VEGF and p53 showed a significant correlation with the histological and nuclear grade of the carcinoma. The data presented in our study indicate that IL-17 might act as a valuable tumor marker in patients with CRC and that combined analysis of p53 and VEGF expression might provide additional information about tumor features.

The cytotoxic effects of some selected gold(III) complexes on 4T1 cells and their role in the prevention of breast tumor growth in BALB/c mice.

PURPOSE: to investigate the cytotoxic activity of newly synthesized gold(III) complexes [AuCl(2)(en)](+), [AuCl(2) (SMC)](+), [AuCl(2)(DMSO)(2)(+) (en: ethylenediamine, SMC: S-methyl- L-cysteine and DMSO: for dimethylsulfoxide) in 4T1 mouse breast cancer cell line in vitro and in vivo and to compare their antitumor characteristics with cisplatin complex [PtCl(2)(NH(3))(2)]. METHODS: the in vitro, effects of the tested complexes on 4T1 cell viability were determined using MTT colorimetric technique. In vivo, progression of mouse breast tumor growth in BALB/c mice was measured by using external caliper. RESULTS: among the tested gold(III) complexes, [AuCl(2) (en)](+) showed best cytotoxic effects in vitro. The cytotoxic effects of [AuCl(2)(en)](+) and [PtCl(2)(NH(3))(2)] were similar at all concentrations. The data from the in vivo experiment showed that among the tested gold(III) complexes only [AuCl(2)(en)](+) can prevent the primary breast tumor growth. [AuCl(2)(en)](+) was tolerated well and much better than [AuCl(2)(DMSO)(2)(+), [AuCl(2)(SMC)](+) and [PtCl(2)(NH(3))(2)] complex which was confirmed by weight gain in mice that received [AuCl(2)(en)](+). In addition, mice that received [AuCl(2)(en)](+) showed better survival time in comparison with mice that received [PtCl(2) (NH(3))(2)] complex. CONCLUSION: [AuCl(2) (en)](+) complex seems to be good candidate for future pharmacological evaluation in breast cancer research.

Choice architecture-based prescribing tool for TB preventive therapy: a pilot study in South Africa.

BACKGROUND: All people with HIV who screen negative for active tuberculosis (TB) should receive isoniazid preventive therapy (IPT). IPT implementation remains substantially below the 90% WHO target. This study sought to further understanding of IPT prescription by piloting a simplified prescribing approach. SETTING: Primary care clinics in Matlosana, South Africa. DESIGN: This was a mixed-methods implementation study. METHODS: Nine providers were recruited and underwent training on 2018 WHO guidelines. A simplified prescribing tool containing antiretroviral therapy (ART) and IPT prescriptions was introduced into the workflow for 2 weeks. Prescription data were collected from file review. Interviews were conducted with prescribers. RESULTS: During the study period, 41 patients were evaluated for ART initiation; 34 (83%) files used the simplified prescribing tool. Thirty-seven (90%) patients were eligible for same-day ART and IPT initiation, of whom 36 (97%) received IPT prescription. Qualitative interviews identified the following barriers to IPT prescription: cognitive burden, extensive documentation, limited management support, paucity of training, stock-outs, and patient-related factors. Provider acceptability of the tool was favorable, with unanimous recommendation to colleagues on the basis of streamlining documentation and reminding to prescribe. CONCLUSIONS: This simplified prescribing device for IPT was feasible to implement. Streamlining documentation and reminding providers to prescribe can reduce work-flow barriers to IPT provision.

Allergen extract-induced interleukin-10 in human memory B cells inhibits immunoglobulin E production.

BACKGROUND: Elevated specific IgE antibody levels are common in atopic individuals, caused by T-helper type 2-dominated B cell activation. The induction of antigen-specific IL-10 secreting T cells is discussed as an important mechanism during specific immunotherapy. By contrast the presence and function of B cell-derived IL-10 is not well defined yet. OBJECTIVE: We investigated whether type-I allergen extracts induce IL-10 expression in human B cells and analysed its functional role on IgE production. METHODS: Human peripheral B cells were stimulated with grass pollen, house dust mite (HDM) (Dermatophagoides pteronyssimus; Der p) and dog allergen extract. Expression of IL-10 by activated human B cells was determined by flow cytometric analysis and ELISA. Functional analysis considering immunoglobulin production was assayed by ELISA. RESULTS: The allergen extracts studied induced IL-10 expression in B cells. However, the ability to induce IL-10 differed between the allergen extracts. The most potent allergen extract was dog (169+/-28 pg/mL), followed by grass pollen (141+/-10 pg/mL) and HDM allergen (125+/-11 pg/mL). Upon allergen extract stimulation only CD27 expressing memory B cells produced IL-10 and co-expressed the very early activation antigen CD69. The addition of allergen extracts to B cells activated by anti-CD40 and IL-4 selectively inhibited IgE which was dependent on allergen extract-induced IL-10. By contrast the other immunoglobulin subclasses like IgA, IgG or IgM were not altered upon allergen extract challenge. CONCLUSION: Our data indicate that allergen-activated memory B cells can modulate IgE production through secretion of IL-10.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation.

BACKGROUND: Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. METHODS: The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. RESULTS: Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. CONCLUSIONS: Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Monosodium glutamate induces apoptosis in naive and memory human B cells.

The aim of this study was to establish the existence of mGluR7 in normal B lymphocytes and analyse the effect of monosodium glutamate (MSG) on B cell apoptosis in vitro. B cells were purified by magnetic cell sorting using anti-CD19-coupled magnetic beads. Cells (10(6)/ml) were cultured with increasing MSG concentrations (1-100 mM). Detection of apoptosis by flow cytometry was performed using the Annexin V-FITC/Propidium iodide (PI) apoptosis detection kit. Naïve and memory B cell population were identified by CD27 staining. Expression of GluRs was determined using PCR. Exposure to increasing MSG concentrations displayed dose dependent effect on B cell viability altogether, ranging from 35% with 100 mM up to 80% with 1 mM MSG. Moreover, the number of late apoptotic cells as well as necrotic cells was dose dependant. Both CD27- as well as CD27+ B cells were affected by MSG. Basal expression of GluRs7 was detected in unstimulated B cells. Glutamate induced apoptosis can be seen in memory as well as naive B cell population and is probably mediated through mGluR7, whose expression in B cells we also confirmed. Our study suggests a new possible mechanism of crosstalk between the nervous and the immune system through glutamate as a potential key mediator (Fig. 4, Ref. 27). Full Text (Free, PDF) www.bmj.sk.

An observational study of safe and risky practices in funeral homes in South Africa.

BACKGROUND: Funeral home personnel are at risk of exposure to infectious hazards. The high prevalence of infectious diseases in South Africa means that these workers and family members of deceased individuals are vulnerable to infection if proper safety measures and equipment are not used. OBJECTIVES:  To collect observational information on funeral industry practices in order to assess the safety of handling corpses and exposure to risk that could result in disease transmission. METHODS: A cross-sectional study was conducted across two locations from August to October 2015. Funeral homes in Klerksdorp and Soweto were approached. The study team did facility assessments and observed preparation practices, focusing on safety equipment, personal protective equipment (PPE) and contact with hazardous materials. Interviews with funeral home personnel and relatives of the deceased were also conducted. RESULTS: Of the funeral homes, 23.0% (20/87) agreed to participate. A median of 5 personnel (interquartile range 4 - 8) were employed per facility. It was observed that not all PPE was used despite availability. Gloves, aprons and face masks were most commonly worn, and no personnel were observed wearing boots, gowns or plastic sleeves. Funeral homes were located near food outlets, schools and open public spaces, and not all had access to proper biohazardous waste disposal services. Of 5 family members who were interviewed for the study, none reported being willing to partake in the funeral preparation procedure. CONCLUSIONS: There is a need to standardise the use of safety equipment, waste disposal methods and location designation in the funeral industry.

Mobile phone access and comfort: implications for HIV and tuberculosis care in India and South Africa.

SETTING: India and South Africa shoulder the greatest burden of tuberculosis (TB) and human immunodeficiency virus (HIV) infection respectively, but care retention is suboptimal.OBJECTIVE: We conducted a study in Pune, India, and Matlosana, South Africa, 1) to identify the factors associated with mobile phone access and comfort of use, 2) to assess access patterns.DESIGN: A cross-sectional study assessed mobile phone access, and comfort; a longitudinal study assessed access patterns.RESULTS: We enrolled 261 participants: 136 in India and 125 in South Africa. Between 1 week and 6 months, participant contact decreased from 90% (n = 122) to 57% (n = 75) in India and from 93% (n = 116) to 70% (n = 88) in South Africa. In the latter, a reason for a clinic visit for HIV management was associated with 63% lower odds of contact than other priorities (e.g., diabetes mellitus, maternal health, TB). In India, 57% (n = 78) reported discomfort with texting; discomfort was higher in the unemployed (adjusted OR [aOR] 4.97, 95%CI 1.12-22.09) and those aged ≥35 years (aOR 1.10, 95%CI 1.04-1.16) participants, but lower in those with higher education (aOR 0.04, 95% CI 0.01-1.14). In South Africa, 91% (n = 114) reported comfort with texting.CONCLUSION: Mobile phone contact was poor at 6 months. While mHealth could transform TB-HIV care, alternative approaches may be needed for certain subpopulations.

Suitability of group-level oral fluid sampling in ruminant populations for lumpy skin disease virus detection.

The geographic expansion of Lumpy skin disease (LSD) from the near East into the European Union highlighted again the need for appropriate disease detection tools applicable to animal host populations where access to individual animals is difficult. This is of particular importance considering that the clinical manifestation of LSD is often mild making early disease detection challenging under the above-mentioned conditions. Building on positive experiences of group-level oral fluid sampling for pathogen detection as it is known to work for swine herds and wild boar, the concept was transferred to ruminants. Two groups of six cattle were infected experimentally with Lumpy skin disease virus (LSDV) under controlled conditions. Blood as well as oropharyngeal and nasal swab samples were collected at regular intervals. Group samples were obtained by placing cotton gauze around a salt lick block provided commonly as dietary supplement. Pieces of the gauze with visible signs of manipulation were tested in parallel to samples obtained from individual animals. Genome load analysis by qPCR technology revealed LSDV detection window starting from day 2 post infection until day 28 post infection, the end of the animal trial. At the individual level, detection periods varied between animals and type of sample and included intermitted detection. The accumulative character of the alternative sampling method makes it suitable to detect LSDV DNA at group-level even at times of the infection where a selective sampling of individuals from a group - as normally done in LSD surveillance - would have most likely failed in the detection.

The laterodorsal tegmentum contributes to behavioral sensitization to amphetamine.

A critical event in the development of behavioral sensitization is a transient increase in excitatory drive to dopamine neurons of the ventral tegmental area (VTA). This is likely to be due, in part, to the ability of drugs of abuse to produce long-term potentiation, expressed as increased AMPA receptor transmission, at excitatory synapses onto VTA dopamine neurons. We investigated the role of the laterodorsal tegmentum (LDT) in behavioral sensitization because LDT neurons provide an important source of excitatory drive to VTA dopamine neurons, through mixed glutamate and cholinergic inputs. To test the role of the LDT in amphetamine sensitization, ibotenic acid or sham lesions of the LDT were performed 1 week before the first of six daily amphetamine injections. When challenged with amphetamine 13 days after the last injection, sham rats expressed sensitization of stereotypy and post-stereotypy locomotor hyperactivity, whereas the latter was attenuated by ibotenic acid lesions of the LDT. To determine whether plasticity occurs in the LDT during amphetamine sensitization, we used a previously developed microdialysis assay in which increased ability of AMPA to activate a pathway serves as a marker for long-term potentiation. Two days after discontinuing repeated saline or amphetamine injections, the responsiveness of LDT-VTA neurons to AMPA was determined by microinjecting AMPA (0.4 nmol) into the LDT and measuring glutamate efflux in the ipsilateral VTA. Glutamate efflux was transiently increased in both groups but a delayed group difference was apparent with relatively higher glutamate efflux in amphetamine rats 30-60 min after AMPA injection. In parallel experiments, dopamine efflux in the nucleus accumbens (NAc) following intra-LDT AMPA declined in saline rats but remained relatively stable in amphetamine rats. Both results suggest relatively greater excitability of the LDT-VTA-NAc pathway after repeated amphetamine treatment. Our results provide the first evidence that neuronal plasticity in the LDT contributes to behavioral sensitization.