Aptamer-quantum dots platform for SARS-CoV-2 viral particle detection by fluorescence microscopy.

The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps. Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein. Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection.

Genetic aspects of Huntington's disease in Latin America. A systematic review.

We aimed to present a systematic review on Huntington's disease (HD) in Latin America (LA). PubMed and LILACS were searched up to March 2015, reporting confirmed HD cases in LA. Case series, cross-sectional, case-control, and prospective studies were included. From 534 communications, 47 were eligible. Population-based studies were not found; minimal prevalence of 0.5-4/100,000 was estimated for Venezuela and Mexico. Geographical isolates were well characterized in Venezuela and in Peru. CAG repeats at HTT gene varied between 7-33 and 37-112 in normal and expanded alleles, respectively. Intermediate alleles were found in 4-10% of controls. Ages at onset and the expanded CAG repeats correlated with r from - 0.55 to -0.91. While haplotype patterns of Venezuelan and Brazilian chromosomes were similar to those observed in Europeans, haplotypes from Peruvian HD patients did not match the same pattern. The limited number of papers found suggests that HD is poorly diagnosed in LA. Minimal prevalence seemed to be halfway between those of Caucasians and Asians. Range of CAG repeats was similar to those of Europeans. Haplotype studies indicate that majority of HD patients might be of Caucasian descent; an Asian origin for some Peruvian patients was proposed.

Prevalencia de anticuerpos anticitoplasma de neutrófilos en pacientes con artritis psoriática / Prevalence of antibodies anticytoplasm of neutrophils in patients with Psoriatic Arthritis

Los anticuerpos anticitoplasma de neutrófilos (ANCA) han cobrado relevancia en distintas patologías. Recientemente se ha reportado un elevado porcentaje (79%) de ANCAp en pacientes con Artropatía Psoriática (APs). Los objetivos de este estudio fueron evaluar la prevalencia ANCAp en pacientes con APs y compararlo con pacientes con artritis reumatoidea (AR), espondilitis anquilosante (EA), psoriasis cutánea (Ps) y controles sanos (CS). Material y métodos: Se incluyeron pacientes consecutivos con APs según criterios CASPAR, AR (criterios ACR 87), EA (criterios de NY modificados); los CS fueron personas de la población general sin antecedentes o evidencias de enfermedades inmunológicas. Se excluyeron pacientes con antecedentes oncológicos, infecciosos, sarcoidosis u otras enfermedades del tejido conectivo y/o vasculitis. Se consignaron datos demográficos, clínicos, radiológicos, antecedentes familiares y terapéutica actual. Se realizaron cuestionarios de actividad de enfermedad y capacidad funcional: BASDAI, BASFI, PASI y HAQ. Se extrajeron muestras de sangre para determinación de ANCA por IFI en etanol que posteriormente fueron confirmadas por IFI en formol. Se realizó además laboratorio general de rutina. Análisis estadístico: Las variables continuas fueron comparadas por ANOVA o test Student y las variables categóricas por Chi-cuadrado o test de Fisher. Resultados: Se incluyeron 148 pacientes (APs = 43, EA = 22, AR = 41, Controles = 38, Psoriasis cutánea = 4). La mediana de edad fue de 52 años (RIQ: 39,5-59), 66% eran mujeres. En el análisis intergrupo, las EA eran más jóvenes y más frecuentemente (87%) de sexo masculino. El resto de los grupos eran comparables para todas las variables demográficas. 57 pacientes mostraron fluorescencia positiva en etanol: AR: 25 (61%), APs: 14 (32,6%), EA: 11 (50%), CS: 6 (15,8%) y Ps: 1 (25%).(AU)

Antibodies ANCA are important diagnostic tools in different diseases. Recently it has been shown that these antibodies can be observed in 79% of patients with Psoriatic Arthritis (PsA). The purpose of our study was to determine the prevalence of ANCA in patients with PsA and compared to patients with Rheumatoid Arthritis (RA), Ankylosing Spondylitis (AS), Psoriasis (Ps) and healthy controls (HC). Material and methods: Consecutive patients with PsA (CASPAR criteria), RA (ACR 87) and AS (New York criteria) were included. HC were people of the general population without evidence of immunological diseases. Patients with a previous history of oncologic, infectious diseases and sarcoidosis were excluded. Demographic, clinical, radiological and therapeutic data were collected. Disease activity and functional capacity were evaluated using validated and specific questionnaires (BASDAI, BASFI, PASI, and HAQ). ANCAs were determined by indirect immunofluorescence (IIF) on ethanol. Then, the positive ones were confirmed by IIF on formol. Student test, ANOVA, Chi square and Fisher exact test were used for Statistical analysis. Results: 148 patients were included (PsA = 43, AS = 22, RA = 41, Ps = 4 y HC = 38). Median age was 52 years (IQR: 39.5 ¹ 59), 66% were women. AS patients were younger and more frequently men. Other variables were comparable between groups. 57 patients exhibited positive ethanol fluorescence: RA: 25 (61%), PsA: 14 (32.6%), AS: 11 (50%), HC: 6 (15.8%) and Ps: 1 (25%). However only 5 patients showed formol fluorescence: AS: 4/22 (ANCAp = 2, ANCAc = 2) y RA: 1/41 (ANCAp). The frequency of positive ANCA was significantly greater in AS vs. RA (p = 0.046).(AU)

Prevalencia de anticuerpos anticitoplasma de neutrófilos en pacientes con artritis psoriática / Prevalence of antibodies anticytoplasm of neutrophils in patients with Psoriatic Arthritis

Los anticuerpos anticitoplasma de neutrófilos (ANCA) han cobrado relevancia en distintas patologías. Recientemente se ha reportado un elevado porcentaje (79%) de ANCAp en pacientes con Artropatía Psoriática (APs). Los objetivos de este estudio fueron evaluar la prevalencia ANCAp en pacientes con APs y compararlo con pacientes con artritis reumatoidea (AR), espondilitis anquilosante (EA), psoriasis cutánea (Ps) y controles sanos (CS). Material y métodos: Se incluyeron pacientes consecutivos con APs según criterios CASPAR, AR (criterios ACR 87), EA (criterios de NY modificados); los CS fueron personas de la población general sin antecedentes o evidencias de enfermedades inmunológicas. Se excluyeron pacientes con antecedentes oncológicos, infecciosos, sarcoidosis u otras enfermedades del tejido conectivo y/o vasculitis. Se consignaron datos demográficos, clínicos, radiológicos, antecedentes familiares y terapéutica actual. Se realizaron cuestionarios de actividad de enfermedad y capacidad funcional: BASDAI, BASFI, PASI y HAQ. Se extrajeron muestras de sangre para determinación de ANCA por IFI en etanol que posteriormente fueron confirmadas por IFI en formol. Se realizó además laboratorio general de rutina. Análisis estadístico: Las variables continuas fueron comparadas por ANOVA o test Student y las variables categóricas por Chi-cuadrado o test de Fisher. Resultados: Se incluyeron 148 pacientes (APs = 43, EA = 22, AR = 41, Controles = 38, Psoriasis cutánea = 4). La mediana de edad fue de 52 años (RIQ: 39,5-59), 66% eran mujeres. En el análisis intergrupo, las EA eran más jóvenes y más frecuentemente (87%) de sexo masculino. El resto de los grupos eran comparables para todas las variables demográficas. 57 pacientes mostraron fluorescencia positiva en etanol: AR: 25 (61%), APs: 14 (32,6%), EA: 11 (50%), CS: 6 (15,8%) y Ps: 1 (25%).

Antibodies ANCA are important diagnostic tools in different diseases. Recently it has been shown that these antibodies can be observed in 79% of patients with Psoriatic Arthritis (PsA). The purpose of our study was to determine the prevalence of ANCA in patients with PsA and compared to patients with Rheumatoid Arthritis (RA), Ankylosing Spondylitis (AS), Psoriasis (Ps) and healthy controls (HC). Material and methods: Consecutive patients with PsA (CASPAR criteria), RA (ACR ’87) and AS (New York criteria) were included. HC were people of the general population without evidence of immunological diseases. Patients with a previous history of oncologic, infectious diseases and sarcoidosis were excluded. Demographic, clinical, radiological and therapeutic data were collected. Disease activity and functional capacity were evaluated using validated and specific questionnaires (BASDAI, BASFI, PASI, and HAQ). ANCAs were determined by indirect immunofluorescence (IIF) on ethanol. Then, the positive ones were confirmed by IIF on formol. Student test, ANOVA, Chi square and Fisher exact test were used for Statistical analysis. Results: 148 patients were included (PsA = 43, AS = 22, RA = 41, Ps = 4 y HC = 38). Median age was 52 years (IQR: 39.5 – 59), 66% were women. AS patients were younger and more frequently men. Other variables were comparable between groups. 57 patients exhibited positive ethanol fluorescence: RA: 25 (61%), PsA: 14 (32.6%), AS: 11 (50%), HC: 6 (15.8%) and Ps: 1 (25%). However only 5 patients showed formol fluorescence: AS: 4/22 (ANCAp = 2, ANCAc = 2) y RA: 1/41 (ANCAp). The frequency of positive ANCA was significantly greater in AS vs. RA (p = 0.046).

Amerindian ancestry in Argentina is associated with increased risk for systemic lupus erythematosus.

Previous studies have demonstrated that in admixed populations, West African ancestry is associated with an increased prevalence of systemic lupus erythematosus (SLE). In the current study, the effect of Amerindian ancestry in SLE was examined in an admixed population in Argentina. The Argentine population is predominantly European with approximately 20% Amerindian admixture, and a very small (<2%) contribution from West Africa. The results indicate that Amerindian admixture in this population is associated with a substantial increase in SLE susceptibility risk (Odds Ratio=7.94, P=0.00006). This difference was not due to known demographic factors, including site of collection, age and gender. In addition, there were trends towards significance for Amerindian ancestry influencing renal disease, age of onset and anti-SSA antibodies. These studies suggest that populations with Amerindian admixture, like those with West African admixture, should be considered in future studies to identify additional allelic variants that predispose to SLE.

No evidence of association between genetic variants of the PDCD1 ligands and SLE.

PDCD1, an immunoreceptor involved in peripheral tolerance has previously been shown to be genetically associated with systemic lupus erythematosus (SLE). PDCD1 has two ligands whose genes are located in close proximity on chromosome 9p24. Our attention was drawn to these ligands after finding suggestive linkage to a marker (gata62f03, Z=2.27) located close to their genes in a genome scan of Icelandic families multiplex for SLE. Here, we analyse Swedish trios (N=149) for 23 single nucleotide polymorphisms (SNPs) within the genes of the PDCD1 ligands. Initially, indication of association to eight SNPs was observed, and these SNPs were therefore also analysed in Mexican trios (N=90), as well as independent sets of patients and controls from Sweden (152 patients, 448 controls) and Argentina (288 patients, 288 controls). We do not find support for genetic association to SLE. This is the first genetic study of SLE and the PDCD1 ligands and the lack of association in several cohorts implies that these genes are not major risk factors for SLE.

Uses and limitations of two molecular cytogenetic techniques for the study of arrested embryos obtained through assisted reproduction technology

We studied chromosomal abnormalities in arrested embryos produced by assisted reproductive technology with fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in order to determine the best technique for evaluating chromosomal aneusomies to be implemented in different situations. We examined individual blastomeres from arrested embryos by FISH and arrested whole embryos by CGH. All of the 10 FISH-analyzed embryos gave results, while only 7 of the 30 embryos analyzed by CGH were usable. Fifteen of the 17 embryos were chromosomally abnormal. CGH provided more accurate data for arrested embryos; however, FISH is the technique of choice for screening in preimplantation genetic diagnosis, because the results can be obtained within a day, while the embryos are still in culture.

Differential expression of CPD1 during postnatal development in the mouse cerebellum.

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.

Comparison of clinical and pathological phenotypes in two ethnically and geographically unrelated pedigrees segregating an equivalent presenilin 1 mutation.

At least 30 different missense mutations have been identified within the presenilin 1 (PS1) gene in pedigrees transmitting familial Alzheimer's disease. The authors investigated the clinical and pathological features of affected members of two pedigrees segregating a PS1 Met146Leu mutation. Genetic relationships between these pedigrees can be effectively excluded on the basis of genealogical data and the fact that although the amino acid substitution is identical, the nucleotide mutations are different. The clinical picture shows remarkable similarities in the neurological and the neuropathological findings between the two pedigrees. This general clinical and pathological concordance argues that much of the disease phenotype arises directly from the effects of the amino acid substitution within the PS1 protein itself. Clinical differences could arise from a direct effect of the difference in base sequence or, alternatively, from the effect of genetic or environmental modifiers.

Nonsurgical correction of nasal deformity in unilateral complete cleft lip: a 6-year follow-up.

Nasal deformity in unilateral cleft lip and palate patients increases with time, tongue malposition being one of the causes. Some authors have emphasized the role of nasal and adjacent facial musculature as active extrinsic agents. Another cause of alar deformity can be the lack of a proper foundation because of a maxillary hypoplasia in the region of the pyriform foramen. If alar collapse occurs, the septum bends convexly toward the cleft side. Tissues are soft and plastic during the neonatal period. Once the infant is about 3 months of age, it becomes difficult to correct the nasal deformity. Therefore, any resource used from the first day, and mainly during the first 15 days of life, will be useful to prevent the increasing deformity and to avoid the surgical correction. A controlled clinical trial was planned to compare the anthropometric measurements of the nasal region in two series of patients with unilateral complete cleft lip. In the first group, we included 44 patients who came to our clinic during the first 2 days of life and the second group consisted of 47 patients who were more than 15 days of age at the time of the first consultation. To provide control data for the evaluation of the results after 6 years of follow-up in both series of cleft patients, we also included a third group of 48 healthy 6-year-old children. A nasal component added to the occlusal prostheses was only used in the first group up to the time of surgery. The same surgeon performed a Millard II procedure with muscular reposition as described by Delaire in all the patients. Nasal measurements taken with a caliper, obtained directly from plaster models by using surface impressions of the babies, were confirmed by a laser three-dimensional measuring device. The statistical comparison between both series showed a significant increase of the columellar length in the first group. A 6-year follow-up to compare growth and cosmetic results of the nose revealed a better and permanent nasal nostril symmetry and no alar cartilage luxation in the patients who had had the nasal component. These results highlight the importance of the early treatment and allow us to suggest the nasal prostheses as a way to prevent the increasing nasal deformity, to help nasal remodeling, to obtain columellar elongation, and to avoid or decrease the need for primary surgery of the cleft nose.

Microcephaly, characteristic facies, joint abnormalities, and deficient leucocyte chemotaxis: a further case of the syndrome of Say et al.

We report on a 13 year old boy with microcephaly, sloping forehead, prominent nose, scoliosis, and flexion contractures involving the elbows and knees. The patient showed severe mental and growth retardation. Since birth and up to the present he has suffered from multiple and varied infections. Immunological studies showed a marked decrease in leucocyte chemotaxis. Clinical and laboratory findings confirm the similarity of this case to the two brothers described by Say et al. We have not found any descriptions of similar patients. The purpose of this paper is to contribute to the phenotypic delineation of this syndrome and to highlight the need for immunological investigation in patients with multiple congenital malformations.

Regulation of differentiation of peritoneal B-1a (CD5+) B cells. Activated peritoneal macrophages release prostaglandin E2, which inhibits IgM secretion by peritoneal B-1a cells.

The B-1a (CD5+) subset of B cells comprises the majority of B cells in the peritoneal cavity and is implicated in the pathogenesis of certain autoimmune diseases and lymphoproliferative disorders. When we stimulated purified B-1a cells with LPS, they produced more than four times as much IgM as similarly stimulated whole peritoneal cells (containing the same number of B-1a cells). Reconstitution experiments using FACS-purified peritoneal cell populations revealed that resident peritoneal macrophages (Mac1+, B220-) profoundly inhibited the LPS response of peritoneal B-1a cells. Culture of B-1a cells with peritoneal macrophages at a ratio of 3:1 (reflecting the in vivo ratio) resulted in a fivefold or greater reduction in the IgM response to LPS. LPS activation of macrophages resulted in production of a soluble factor that inhibited LPS-induced B cell differentiation by 86% when used at a concentration of 5%. When [3H]arachidonic acid-pulsed macrophages were stimulated with LPS, the major arachidonic acid metabolite secreted was PGE2 (a potent inhibitor of B cell differentiation). The inhibitory capacity of the macrophage-derived supernatant was reversed by the addition of anti-PGE2. These findings indicate that macrophage-derived PGE2 functions as an important regulator of polyclonal response of B-1a cells to LPS.

Regulation of apoptosis in vitro in mature murine spleen T cells.

Previously it has been shown that thymocytes undergo apoptosis, a form of programmed cell death, in response to glucocorticoids. This classic form of apoptosis is prevented by inhibition of protein synthesis. The current paper demonstrates that mature T cells also undergo apoptosis, but that the regulation of apoptosis in spleen T cells differs from that of thymocytes. Mature mouse spleen T cells were shown to die by apoptosis, not necrosis, when cultured without an added stimulus. Assays for apoptosis included internucleosomal DNA cleavage by gel electrophoresis, percent fragmentation of DNA by the diphenylamine method, and percent of cells with hypodiploid DNA by flow cytometry. The percent of apoptotic cells was 2% in fresh spleen T cells, and increased at least until 16 h, when 21% were apoptotic. Dexamethasone caused apoptosis in both thymus and spleen T cells, but only thymocytes showed a requirement for protein synthesis in dexamethasone-induced death. Cycloheximide increased apoptosis in spleen T cells, indicating that apoptosis was controlled by newly synthesized protective proteins. Spontaneous apoptosis was decreased in spleen T cells by protein kinase C activation, and was increased by H7 and staurosporine, which inhibits protein kinases, in contrast with the behavior of thymocytes. The protein kinase A/G inhibitor HA1004 also decreased spleen T cell apoptosis. The contrasting effects of cycloheximide on thymocytes and spleen T cells occurred over the same concentration range, and the same was true for PMA. The dexamethasone dose-response curves were similar, except that a greater proportion of spleen T cells were dexamethasone-resistant. These data support the hypothesis that the apoptosis program in T cells undergoes a transition during their maturation, such that apoptosis in mature T cells is regulated more like that of mature B cells than that of thymocytes.

Apoptosis in splenic B lymphocytes. Regulation by protein kinase C and IL-4.

Small dense splenic B lymphocytes from adult specific pathogen-free mice were shown to undergo apoptosis in vitro as indicated by internucleosomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically from < 2 to 32% of B cells by 16 h. The rate of accumulation of apoptotic cells was reduced by the addition of IL-4 or PMA, but not by the inactive phorbol ester, 4 alpha PDD. In contrast, inhibitors of protein kinase C (H7 and staurosporine) increased the percentage of cells undergoing apoptosis to > 70% by 12 h; HA 1004, genistein, and herbimycin A all had no effect on apoptosis. Thus, protein kinase C activity regulates apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, indicating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic cells measured by flow cytometry and the percentage of fragmented DNA measured by the diphenylamine method were nearly equal, regardless of the method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apoptotic, these results suggest that in individual B cells apoptosis progresses rapidly to completion. These data suggest a fundamental change in our concept of the life-style of the "resting" B cell: instead of a dormant cell remaining unchanged until it receives activation signals, the mature spleen B cell appears programmed to die by apoptosis unless rescued by specific agents, such protein kinase C activators or IL-4.