Research advances on polyglycine diseases / 中华神经科杂志

Nucleotide repeat expansion is one of the common causes for neurodegenerative disorders. Polyglycine diseases are a newly defined neuro- and muscle- degenerative disease spectrum characterized by CGG trinucleotide repeat expansions, generation and aggregation of aberrant polyglycine protein, and formation of intranuclear inclusions. To date, the aggregation of pathogenic polyglycine protein has been proved in fragile X-associated tremor/ataxia syndrome and neuronal intranuclear inclusion disease. In recent years, the case load of these diseases grows rapidly with the increasing awareness and developing genetic testing technologies. This article aims to systematically review the recent progress in polyglycine diseases, and probe into their pathogenic mechanisms as well as clinical concerns.

Inclusión body myositis: A differential diagnosis to consider in patients with myopathy refractory to immunosuppressants. Two case reports / Miopatía por cuerpos de inclusión: un diagnóstico diferencial que considerar en pacientes con miopatía refractaria a inmunosupresores. Reporte de 2 casos

ABSTRACT Inclusion body myositis is part of the group of inflammatory myopathies, representing 30% of this group of diseases, and is considered an orphan disease because its estimated prevalence is less than 5 per 10,000 inhabitants. It produces weakness and atrophy of the proximal and distal muscles. The pathophysiological mechanisms are mainly autoimmune, inflammatory, and degenerative. The cases are presented of two female patients who came to : the emergency department due to progressive loss of upper and lower limb strength, and progressive asymmetric muscle weakness.

RESUMEN La miositis por cuerpos de inclusión forma parte del grupo de las miopatías inflamatorias, de las que representa el 30%; es considerada una enfermedad huérfana, ya que se estima que su prevalencia es menor a 5 por cada 10.000 habitantes. Produce debilidad y atrofia de los músculos proximales y distales. Los mecanismos fisiopatológicos son principalmente autoinmunes, inflamatorios y degenerativos. Se presentan 2 casos de mujeres, quienes acudieron a urgencias por pérdida progresiva de la fuerza en miembros superiores e inferiores, con debilidad muscular asimétrica de curso progresivo.

Inclusões semelhantes aos corpúsculos de Russell em blastos mieloides / Inclusions similar to Russell bodies in myeloblasts

Alterações nos mecanismos celulares resultam no aparecimento de corpúsculos de Russel, que são inclusões intracitoplasmáticas basofílicas. Estas inclusões ocorrem devido a uma indigestão celular de proteínas e podem ser observadas em diversas condições patológicas como leucemias, mieloma múltiplo, linfoma de Burkitt e gastrite com corpúsculos de Russell, sendo raramente observadas em leucemias mieloides agudas. O presente estudo relata a presença de inclusões semelhantes aos corpúsculos de Russell em blastos no sangue periférico de uma paciente de 51 anos com leucemia mieloide aguda.

Changes in cell mechanisms result in the appearance of Russel bodies, which are basophilic intracytoplasmic inclusions. These inclusions occur due to cellular indigestion of proteins, and can be observed in several pathological conditions such as leukemias, multiple myeloma, Burkitt's lymphoma, and gastritis with Russell bodies, but rarely observed in acute myeloid leukemias. The present study reports the presence of inclusions similar to Russell bodies in peripheral blood blasts in a 51-year-old female patient with acute myeloid leukemia.

Miopatías autoinmunes: revisión de diagnóstico y manejo / Autoimmune myopathies: diagnosis and management review

Las miopatías inflamatorias idiopáticas (MII) son un grupo de enfermedades autoinmunes crónicas que afectan principalmente a los músculos proximales. Los tipos más comunes son dermatomiositis (DM), polimiositis (PM), miopatía autoinmune necrotizante y miositis por cuerpos de inclusión. Se identifican de forma única por su presentación clínica que consiste en manifestaciones musculares y extramusculares, sus alteraciones miopáticas en el electromiograma y la elevación de las enzimas musculares. Sin embargo, la biopsia muscular sigue siendo el gold estándar para el diagnóstico. Estos trastornos son potencialmente tratables con un diagnóstico adecuado. Los objetivos del tratamiento son eliminar la inflamación, restaurar el rendimiento muscular, reducir la morbilidad y mejorar la calidad de vida. Esta revisión tiene como objetivo proporcionar un enfoque de diagnóstico básico a los pacientes con sospecha de MMI a través de sus principales hallazgos clínicos, de laboratorio e histopatológicos.

Idiopathic inflammatory myopathies (MII) are a group of autoimmune diseases that mainly affect the proximal muscles. The most common types are Dermatomyositis (DM), Polymyositis (PM), Necrotizing autoimmune myopathy and Inclusion body myositis. Unique forms are identified in their clinical presentation consisting of muscular and extramuscular manifestations, their myopathic alterations in the electromyogram and the elevation of muscle enzymes. However, muscle biopsy remains the gold standard for diagnosis. These disorders are tratable with a proper. The goals of treatment are to eliminate inflammation, restore muscle performance, reduce morbidity and improve quality of life.This review aims at a basic diagnostic approach in patients with suspicion of MMI through its main clinical, laboratory and histopathological findings.

Ultrastructural and clinical findings of mitochondrial encephalomyopathy:report of 27 cases / 中华病理学杂志

Objective@#To investigate the ultrastructural features of muscle in patients with mitochondrial encephalomyopathy for its diagnosis and differential diagnosis.@*Methods@#The clinical data of 27 mitochondrial encephalomyopathy patients who underwent left or right biceps brachii muscle biopsy at Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University from July 2006 to August 2017 were analyzed retrospectively. The muscle biopsy specimens were examined underlight microscope and transmission electron microscope.@*Results@#There were 27 patients (17 males, 10 females) with an age range of 12 to 62 years (mean 29 years). The age of onset ranged from 3 to 38 years. The course of disease ranged from 1 month to 24 years. Twenty-two cases presented with lactic acidosis and stroke-like episodes (MELAS) syndrome, four with myoclonic epilepsy with ragged red fibers (MERRF) syndrome, and one with chronic progressive paralysis of extraocular muscle (CPEO) syndrome. Skeletal muscle biopsy showed abundant ragged red fibers and strongly SDH-reactive vessel. Genetic studies showed 17 of 22 cases of MELAS syndrome had A3243G mutation, and the other 5 cases had no abnormality. A8344G mutation was found in 3 of 4 cases of MERRF syndrome. No single or multiple mtDNA mutations were found in the single case of CPEO. Transmission electron microscopy of all 27 cases showed diffuse proliferation of mitochondria between the myofibrils and beneath the sarcolemma, with increased spacing between muscle cells. Seven cases showed numerous glycogen and four showed subsarcolemmal lipid droplets, 13 cases showed unusual mitochondrial morphology, including mitochondrial electron-dense substances and paracrystal line inclusions ("parking lot" change)in eight cases.@*Conclusions@#Transmission electron microscopy shows significant differences in ultrastructural pathological changes among different patients with mitochondrial encephalomyopathy. Some patients with mild clinical symptoms have increased mitochondrial number, increased metabolism of glycogen and lipid droplets, while others with severe clinical symptoms have abnormal mitochondrial morphology. Typical crystalloid inclusions are found in mitochondria, which are of great value in the diagnosis of this disease.

Cytomegalovirus infection with skin lesions as the primary manifestation: a case report / 中华皮肤科杂志

A case of cytomegalovirus infection with skin lesions as the primary manifestation is reported.A 46-year-old female patient presented with a 3-month history of painful perioral blisters and erosions,and a 6-week history of erythema,blisters and erosions on the left ann.The patient was ever diagnosed with systemic lupus erythematosus and lupus nephritis 12 years prior to the presentation.Systemic lupus erythematosus was exacerbated 5 months prior to this presentation,and glucocorticoids and mycophenolate mofetil were administered.Skin examination revealed irregularly shaped perioral blisters with erosions and crusts,localized patchy erythema with erosion in the center on the flexor aspect of the left forearm,erythema and blisters on the left upper arm,and multiple petechiae and ecchymoses on the abdomen.Histopathological examination of the skin lesion on the left upper limb showed epidermal necrolysis with scattered viral inclusion bodies.Immunohistochemical examination revealed positive staining for cytomegalovirus antigen in giant cells in the necrolytic epidermis.Cytomegalovirus DNA was detected in exudates from lesions.However,cytomegalovirus DNA was not detected in the serum in the initial test,but became positive (8.04 × 103 copies/ml) 1 week later.In addition,anti-cytomegalovirus IgG antibodies were detected in the serum.The patient was diagnosed with cutaneous cytomegalovirus infection.Affter the treatment with both oral and topical ganciclovir,the lesions were improved gradually,followed by severe pulmonary infection,and the patient was finally died of multiple organ failure.

Fatal Disseminated Herpes Simplex in a very premature neonate

Herpes Simplex Virus infections (HSV) are ubiquitous. The neonatal HSV infection (NHSV) is rare. The incidence is estimated globally at only 10.3 per 100,000 births, but it can cause devastating disease in premature infants. Both HSV-1 and HSV-2 can be the etiologic agents in this type of vertically transmittted NHSV infection. Here we describe the pathological findings from a complete autopsy of a very low birth weight infant who succumbed to the infection despite early institution of antiviral treatment. We urge more awareness of this disease with continued surveillance; every effort should be taken to make an early diagnosis and thus prevent this devastating disease.

Construction and identification of the recombinant M13-IN5 phage and its effect on Chlamydia trachomatis / 中华皮肤科杂志

Objective To construct active phages against Chlamydia trachomatis,and to evaluate its effect on Chlamydia trachomatis.Methods The M13 phage was recombined with the IN5 sequences encoding the capsid protein VP1 of chlamydiophage phiCPG1,and then the recombinant M13-IN5 phage was obtained.PCR amplification,enzyme digestion and sequencing were performed to verify whether the target fragment was inserted into the phage successfully.The viability of the phage was evaluated by plaque formation assay.Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of M13 phage and recombinant M13-IN5 phage at the titer of 1011 plaque-forming units (PFU)/ml on the proliferation of Hela cells,and Hela cells uninfected with chlamydia served as the blank control group.Western blot analysis was performed to determine the expression of the IN5 loop protein in the recombinant M13-IN5 phage,M13 phage and Escherichia coli ER2738 at exponential growth phase.Cultured standard Chlamydia trachomatis serovar E strain was treated with M13 phage and recombinant M13-IN5 phage at the titer of 1011 PFU/ml separately,and chlamydia control group without the treatment with phages was set up.After 36-hour infection,confocal microscopy was performed to detect the location of the M13 phage and the recombinant M13-IN5 phage.Moreover,iodine staining was conducted to count inclusion bodies at 36,48,60 and 72 hours separately after infection.Statistical analysis was carried out by a two-sample t-test for comparisons between two groups,one-way analysis of variance (ANOVA) for intergroup comparison,and Bonferroni test for multiple comparisons.Results The bioactive recombinant M13 phage containing the IN5 loop gene was constructed successfully,and Western blot analysis confirmed that the recombinant phage expressed IN5 loop/p Ⅲ fusion protein with a high titer of 3.05 × 1011 PFU/ml.As CCK8 assay showed,there was no significant difference in proliferation of Hela cells among the blank control group,M 13 phage group and recombinant M13-IN5 phage group (A450 values:3.63 ± 0.01,3.55 ± 0.02,3.70 ± 0.01,respectively,F =12.0,P ＞ 0.05).Confocal microscopy showed overlap between the phage fluorescence and chlamydial inclusion body fluorescence.The M13-IN5 phage group and M13 phage group both showed significantly decreased number of inclusion bodies compared with the control group (both P ＜ 0.05) at 36 and 72 hours after chlamydial infection,and the number of inclusion bodies was significantly lower in the M 13-IN5 phage group than in the M13 phage group (P ＞ 0.05).After 48,and 60 hours of chlamydial infection,the number of inclusion bodies did not differ among the M13 phage group,M13-IN5 phage group and control group (both P ＞ 0.05).Conclusions The recombinant M13-IN5 phage was bioactive and could successfully express the IN5 loop protein.In the in vitro experiments,the recombinant phage could enter into chlamydia inclusion bodies,and markedly inhibited the infection of Chlamydia trachomatis.

Diseño y producción de diversas proteínas fusión de la nicotinamida/ nicotinato mononucleotido adenilil transferasa (NMNAT) de Plasmodium falciparum / Design and production of various fusion proteins of the nicotinamide/ nicotinate mononucleotide adenilil transferase (NMNAT) of Plasmodium falciparum / Desenho e produção de várias proteínas fusão da nicotinamida/nicotinato mononucleotido adenilil transferasa (NMNAT) do Plasmodium falciparum

Resumen Las proteínas recombinantes se han convertido en herramientas útiles en la investigación bioquímica. Sin embargo, durante su producción, aparecen cuerpos de inclusión (IB), debido, por un lado, a la alta expresión de proteína producida a partir de los vectores usados que poseen promotores de alta eficiencia y, por otro lado, a características propias de la proteína. Ahora bien, la nicotinamida/nicotinato mononucleótido adenililtransferasa (NMNAT) es una proteína central en la biosíntesis del NAD(H)+, molécula esencial en el metabolismo celular, y ha sido estudiada en parásitos protozoos. Para el estudio de la NMNAT de estos parásitos se ha recurrido a la expresión de su versión recombinante en E. coli, obteniéndose gran cantidad de proteína como IB. Con el fin de aumentar la solubilidad de la proteína, se clonó la secuencia codificante de la NMNAT de Plasmodium falciparum en diferentes vectores de expresión, se indujo la expresión de la proteína recombinante en E. coli BL21 (DE3) y se analizó la solubilidad. La proteína fusión con mayor solubilidad fue purificada y evaluada enzimáticamente. La adición de la etiqueta MBP (proteína de unión a maltosa) a la PfNMNAT incrementó su solubilidad y permitió obtener una proteína funcional con una alta pureza.

Abstract Recombinant proteins have become useful tools in biochemistry research. During their production, however, inclusion bodies (IB) appear, on the one hand, due to the high expression rate from the recombinant plasmids, which have high efficiency promoters, and, on the other hand, intrinsic characteristics of the expressed protein. Furhtermore, the nicotinamide/nicotinate mononucleotide adenilyl transferase (NMNAT) is a central protein in NAD(H)+biosynthesis, an essential cofactor in cell metabolism, and in protozoon parasite has been studied. To study the NMNAT protein of these parasites, their recombinant version in E. coli has been expressed, getting a great quantity of IB as a by-product. To increase the solubility of the protein, the coding sequence of the NMNAT enzyme of Plasmodium falciparum was cloned in different expression plasmids which were subsequently transformed into E. coli BL21 (DE3) expression strain. The solubility of the recombinant proteins was assessed and the one with the highest presence in the soluble fraction was subsequently purified and its enzyme activity was determined. The recombinant protein with a MBP (maltose-binding protein) tag showed an increased solubility and purity.

Resumo As proteínas recombinantes tem convertido em ferramentas úteis na investigação bioquímica. Um problema frequente durante a sua produção é a aparição de corpos de inclusão (IB), devida principalmente à elevada expressão da proteína produzida a partir dos vectores utilizados que têm promotores de elevada eficiência; bem como características das próprias proteínas. Além disso, a nicotinamida/nicotinato mononucleótido adenililtransferasa é uma proteína central na biossíntese do NAD(H)+, molécula essencial no metabolismo celular, e tem sido estudada em protozoários parasitos. Para o estudo da NMNAT destes parasitos, foi realizada a expressão da sua versão recombinante em E. coli, e foi obtido uma grande quantidade de proteína em forma de IB. Com a finalidade de aumentar a quantidade de proteína na fração solúvel, a sequência codificadora para NMNAT de Plasmodium falciparum foi clonada em diferentes vectores de expressão, foi induzida a expressão da proteína recombinante em E. coli BL21(DE3) e foi analisada a solubilidade. A proteína fusão com maior solubilidade foi purificada e avaliada enzimáticamente. A adição da etiqueta MBP (proteína de união a maltosa) à PfNMNAT incrementou a solubilidade da proteína e permitiu obter uma proteína funcional com una alta pureza. espécie de planta.

Lafora and Trétiakoff: the naming of the inclusion bodies discovered by Lewy / Lafora e Trétiakoff: a denominação dos corpos de inclusão descobertos por Lewy

ABSTRACT Fritz Heinrich Jakob Lewy described, for the first time, in 1912, novel peculiar inclusions in neurons of certain brain nuclei in patients with Paralysis agitans, and compared his finding to the amyloid bodies described by Lafora one year before. Gonzalo Rodriguez Lafora studied one patient with Paralysis agitans, in 1913, and recognized, described, and depicted structures identical to those previously reported by Lewy. He was the first to acknowledge Lewy's finding, and also the first to name such inclusions after the discoverer - cuerpos intracelulares de Lewy (Lewy bodies). Konstantin Nikolaevich Trétiakoff named the inclusions he found in neurons of the substantia nigra of patients with Parkinson's disease as corps de Lewy (Lewy bodies), in 1919. Trétiakoff has unanimously received the credit for the eponym. However, Lafora's earlier description should make him deserving of the authorship of the eponym.

RESUMO Fritz Heinrich Jakob Lewy descreveu pela primeira vez, em 1912, inclusões singulares inéditas em neurônios de certos núcleos do cérebro em casos de Paralysis agitans e comparou seu achado aos corpos amilóides, como descrito por Lafora um ano antes. Gonzalo Rodriguez Lafora estudou um caso de Paralysis agitans, em 1913,e reconheceu, descreveu e representou estruturas idênticas às recentemente relatadas por Lewy. Foi o primeiro a reconhecer o achado de Lewy e também o primeiro a denominar tais inclusões segundo seu descobridor - cuerpos intracelulares de Lewy (corpos de Lewy). Konstantin Nikolaevich Tretiakoff designou as inclusões que encontrou em neurônios da substantia nigra em casos de doença de Parkinson de corps de Lewy (corpos de Lewy), em 1919. Ele recebeu o crédito pelo epônimo de modo unânime. Entretanto, a descrição anterior de Lafora deveria fazê-lo merecedor da autoria do epônimo.

Lewy and his inclusion bodies: Discovery and rejection / Lewy e seus corpos de inclusão: descoberta e rejeição

ABSTRACT Fritz Jacob Heinrich Lewy described the pathology of Paralysis agitans [Parkinson disease] and was the first to identify eosinophilic inclusion bodies in neurons of certain brain nuclei, later known as Lewy bodies, the pathological signature of the Lewy body diseases. In 1912, he published his seminal study, followed soon after by an update paper, and 10 years later, in 1923, by his voluminous book, where he exhaustively described the subject. The publication provided extensive information on the pathology of Paralysis agitans, and the entirely novel finding of eosinophilic inclusion bodies, which would become widely recognized and debated in the future. His discovery was acknowledged by important researchers who even named the structure after him. However, after his last publication on the issue, inexplicably, he never mentioned his histopathological discovery again. Despite several hypotheses, the reasons that led him to neglect (reject) the structure which he so preeminently described have remained elusive.

RESUMO Fritz Jacob Heinrich Lewy descreveu a patologia da Paralysis agitans [doença de Parkinson] e identificou pela primeira vez corpos de inclusão eosinófílos em neurônios de certos núcleos cerebrais, conhecidos mais tarde como corpos de Lewy, assinatura patológica das doenças dos corpos de Lewy. Ele divulgou em 1912 seu trabalho seminal, seguido logo por um artigo de atualização e 10 anos depois, em 1923, seu volumoso livro onde detalhou exaustivamente o assunto. Ali ele trouxe extensa informação sobre a patologia da Paralysis agitans e um achado inteiramente novo, os corpos de inclusão eosinófilos, que seriam valorizados e largamente debatidos no futuro. Seu achado foi reconhecido por importantes pesquisadores que até designaram essa estrutura com seu nome. Entretanto, após sua última publicação sobre o assunto, inexplicavelmente , ele nunca mais mencionou sua descoberta histopatológica. Apesar de diversas hipóteses, a razão que o levou negligenciar (rejeitar) a estrutura, que teve a primazia de descrever, permaneceu desconhecida.

Identification of cell invasion-related virulence genes in chlamydial plasmids in vitro / 中华皮肤科杂志

Objective To compare the infectivity of several transformed Chlamydia trachomatis (Ct) mouse pneumonitis (Mopn) strains to host cells, and to identify cell invasion-related virulence genes in Chlamydial plasmids. Methods Several Ct strains, including wild-type Ct Mopn strain(WT strain), plasmid-free Ct strain(CMUT3 strain), Ct Mopn strain transformed with the shuttle vector carrying pGFP and the complete C. muridarum (CM) plasmid (pGFP::CM strain)and Ct Mopn strains transformed with shuttle vectors carrying pGFP and mutant CM plasmids with in-frame deletions of Pgp3, 4, 5 or 7 (pGFP::CM△Pgp3, 4, 5, 7 strains), were cultured, amplified and collected. After the concentrations of Ct were determined, each of these strains was divided into four groups to be inoculated at a same amount(1.5 × 104 inclusion forming units(IFU)) followed by four different treatments respectively: centrifugalization +DEAE group treated with centrifugalization followed by ion-exchange chromatography on diethylaminoethyl(DEAE)-cellulose columns, centrifugalization group treated with centrifugalization only, DEAE group treated with chromatography on DEAE-cellulose columns only, control group receiving no treatment. After additional culture for 20 - 24 hours, indirect immunofluorescence assay was performed to count the number of chlamydial inclusions. At 20, 40 and 60 hours after infection, the growth rate and area of chlamydial plaques were assessed after three continuous passages. Lugol′s iodine staining was conducted to observe glycogen synthesis in bacterial inclusions. Results The inclusion number in the centrifugalization + DEAE group, centrifugalization group, DEAE group and control group was 10.20 ± 1.30, 6.80 ± 0.44, 3.00 ± 1.22 and 0.80 ± 0.45 respectively for the pGFP::CM△Pgp4 strain, 6.40 ± 0.89, 3.80 ± 0.83, 1.60 ± 0.89 and 0.60 ± 0.54 respectively for the CMUT3 strain. Under same experiment conditions, the pGFP::CM△Pgp4 strain and CMUT3 strain showed similar infectivity, and formed less inclusions compared with the other Ct strains (all P < 0.01). The number of inclusions formed by the same Ct strains were significantly different among the 4 groups(F = 845.310, P <0.01), and were highest in the centrifugalization + DEAE group for all the strains. The pGFP::CM△Pgp4 strain showed significantly lower growth rate and area of plaques with an abnormality in glycogen accumulation compared with the other strains at 20, 40 and 60 hours after infection. Conclusion The plasmid-encoding gene Pgp4 may be a cell invasion-associated virulence gene in chlamydial plasmids.

Fibroma digital celular: un tumor periungueal CD34 positivo / Cellular digital fibroma: distinctive CD34 positive tumor

El fibroma digital celular consiste en una neoplasia benigna, generalmente solitaria, localizada en un dedo del pie o de la mano, que suele comprometer la región ungular y regresar espontáneamente. Presentamos un caso de sexo masculino de 27años, con una lesión nodular en el segundo dedo del pie derecho. La histopatología mostró una proliferación fuso celular dérmica acral, con presencia de fibroblastos reactivos y mastocitos, entre haces de colágeno engrosados y capilares dilatados. Inmunomarcación: vimentina (+), CD68 (+) en células aisladas, proteína s100 (-), actina muscular lisa (-), CD34 (+) focal.

Digital fibromas are common benign acral tumors typically reported as angiofibromas (AFs) or acquired digital fibrokeratomas (ADFs). Cellular variants are not well recognized. Nodules develop mainly in fingers and toes. There is high recurrence but spontaneous regression without treatment has also been reported. We report a man of 27 years-old, showing a one cm nodule in the second finger of the right foot. Histological examination revealed spindled tumor cells containing characteristic intracytoplasmic inclusion bodies that stained with vimentin, CD68 positive, protein S100 negative, muscle specific actin negative and CD34 positive. These findings suggest that a subset of digital fibromas is characterized by a dense cellular proliferation of CD34 positive spindle cells. Awareness of this variant of digital fibroma and its staining pattern is critical in preventing misdiagnosis as dermatofibrosarcoma protuberans, particularly in superficial biopsies.

Producción y purificación de anticuerpos aviares (IgYs) a partir de cuerpos de inclusión de una proteína recombinante central en el metabolismo del NAD+ / Production and purification of avian antibodies (IgYs) from inclusion bodies of a recombinant protein central in NAD+ metabolisim / Produção e purificação de anticorpos aviários (IgYs) empregando os corpos de inclusão de uma proteína recombinante importante para o metabolismo do NAD+

El uso de gallinas para la producción de anticuerpos policlonales, reduce la intervención animal, obteniendo además una gran cantidad de anticuerpos. Las aves presentan mayor distancia filogenética entre sus antígenos y los de mamíferos ofreciendo altos porcentajes de anticuerpos específicos. La producción de anticuerpos requiere antígenos en cantidades considerables, por ello es creciente el uso de proteínas recombinantes para tales fines. No obstante, la obtención de proteínas en sistemas heterólogos conduce frecuentemente a la precipitación en agregados insolubles de limitada utilidad (cuerpos de inclusión). Este trabajo presenta una metodología para la producción de anticuerpos policlonales (IgYs) empleando cuerpos de inclusión (CI) como antígeno. Los CI purificados e inoculados corresponden a la Nicotinamida / Nicotinato Mononucleotido Adenililtranferasa (His-GiNMNAT) de Giardia intestinalis expresada en Escherichia coli. La purificación del antígeno se llevó a cabo mediante solubilización y renaturalización. Los anticuerpos se purificaron de la yema de huevo de gallinas imunizadas mediante dilución en agua, seguida de precipitación con sulfato de amonio al 60% y afinidad tiofilica. Los anticuerpos fueron evaluados mediante inmunoblot empleando la proteína His-GiNMNAT. De una yema de huevo se obtuvieron 14,4 mg de IgYs, con alta pureza y con un reconocimiento de hasta 15ng de His-GiNMNAT. Se mejoró la especificidad de los IgYs mediante una purificación adicional por afinidad al antígeno, lo cual permitiría su empleo para el reconocimiento de la proteína del parásito.

In contrast with other animal models, the use of hens for polyclonal antibodies production not only reduces animal intervention, but also increases the quantity of the obtained immunoglobulins. The phylogenetic distance between birds and mammals, leads to more specific avian antibodies than their mammalian counterparts. Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems, frequently leads to the formation of insoluble and useless aggregates (inclusion bodies, IC). This article presents a strategy to produce avian polyclonal antibodies (IgYs) from IC. In order to obtain the antigen, the Giardia intestinalis nicotinamide mononucleotide adenylyltransferase recombinant protein (His-GiNMNAT) was expressed in Escherichia coli. The His-GiNMNAT protein was purified through IC solubilization and re-folding. The purified protein was use to immunize hens. The antibodies were purified from egg yolk by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography. Specificity of the purified antibodies was tested against the His-GiNMNAT protein through Western blot essays. In terms of yield, 14.4 mg of highly pure IgYs were obtained from one egg yolk; these antibodies were able to detect 15 ng of antigen. IgYs specificity was improved by means of antigen affinity purification, allowing its implementation for endogenous detection of GiNMNAT protein in G. intestinalis.

A utilização de galinhas para a produção de anticorpos policlonais, diminui a intervenção sobre o animal e permite a geração de grandes quantidades de anticorpos. As aves têm maior distância filogenética entre os seus antígenos em comparação com os mamíferos, oferecendo altos porcentagens de anticorpos específicos. A produção de anticorpos requerem quantidades consideráveis de antigénio, por conseguinte, é comum a utilização de proteínas recombinantes para esta finalidade. No entanto, a produção de proteínas em sistemas heterólogos muitas vezes leva à precipitação em agregados insolúveis de utilidade limitada (corpos de inclusão). Este trabalho apresenta uma metodologia para a produção de anticorpos policlonais, utilizando proteína recombinante a partir de corpos de inclusão. O antígeno utilizado foi a proteína Nicotinamida / Nicotinato Mononucleótido Adenililtranferasa de Giardia intestinalis gerada em Escherichia coli (His-GiNMNAT). A purificação do antigénio foi feita por solubilização e renaturalização. Os anticorpos foram purificados a partir da gema de ovo de galinhas imunizadas pelo método de diluição em água, seguido de precipitação com sulfato de amónio (60%) e afinidade tiofilica. Os anticorpos foram avaliados por imunoblot utilizando a proteína His-GiNMNAT. De uma gema de ovo foram obtidos 14,4 mg de IgYs com pureza elevada. Estes anticorpos podem reconhecer até 15 ng de His-GiNMNAT. A especificidade dos IgYs foi reforçada por outra purificação por afinidade pelo antigénio. Isto irá permitir a sua utilização para o reconhecimento da proteína do parasita.

Recognition of Russell bodies and Mott cells / 中华检验医学杂志

There are not yet uniform for the recognition and morphological description of Russell bodies and Mott cells.Some difficulties for the learner in clinical diagnosis were encountered.The characteristics of name and morphology of Russell bodies and Mott cells were described in this paper combined with many years of experience in clinical practice according to new point of view of (2008) World Health Organization classification of tumours of haematopoietic and lymphoid tissues and European Leukemia Net Morphology Faculty.

Clinical, pathological and molecular study of two Chinese families with oculopharyngodistal myopathy / 中华神经科杂志

ObjectiveTo investigate the clinical,myopathological and molecular changes in two Chinese families with oculopharyngodistal myopathy ( OPDM).MethodsWe performed muscle biopsy and histopathologic study on the probands of two families,and further examined molecular genetic testing on PABPN1 and GNE gene. Results Family 1 included 3 affected brothers in the same generation and family 2 involved 4 patients in 2 generations. Dysarthria rather than external ophthalmoplegia was the prominent oculopharyngeal symptoms for Chinese patients. No intranuclear inclusions were observed in ultrastructural examination.The number of GCG repeats in the PABPN1 gene was within normal range and no mutations were identified in the GNE gene.ConclusionsFamily 1 is the first publication on autosomal recessive OPDM in China.The age of onset of two families was comparable with Japanese patients and the pattern of muscle involvement was different. OPDM is a distinct phenotypical,histological,and genetic entity.

Cytologic Features of Giant Cell Ependymoma: A Case Report and Review of the Literature

Here, we present a case of anaplastic giant cell ependymoma (GCE) occurring in a 15-year-old woman. Squash smear slides for intraoperative frozen section diagnosis revealed oval to round cell clusters with a papillary structure in a fibrillary background. This was occasionally accompanied by the presence of bizarre pleomorphic giant cells with hyperchromatic nuclei and prominent intranuclear inclusions. These intranuclear inclusions were a key clue to diagnosis of ependymoma. Histologic analysis revealed features of a high-grade tumor with perivascular pseudorosettes and bizarre pleomorphic giant cells, which established the diagnosis of GCE. We performed a review of literatures about the cytologic features of GCE, including our case, thus proposing that intraoperative frozen diagnosis of GCE would be established by squash smear preparations featuring the mitosis and necrosis, as well as the high cellularity, and the presence of giant cells showing hyperchromatic nuclei with eosinophilic cytoplasm and intranuclear inclusions/pseudoinclusions.

Molecular modeling and conformational analysis of native and refolded viral genome-linked protein of Cardamom mosaic virus.

The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5’ end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.

Origin of ovarian epithelial inclusions and its relationship with the development of low-grade serous carcinoma / 中华妇产科杂志

Objective To investigate the possible origin of ovarian epithelial inclusions and its relationship with the low-grade ovarian serous carcinoma.Methods By comparatively evaluating the morphologic (secretory and ciliated cell distribution ) and immunophenotypic [using paired box gene 8 (PAX8),tubulin,calretinin,and Ki-67 as first antibodies] attributes of ovarian epithelial inclusions,the normal tubal epithelium,and the ovarian tumors,all adnexal tissues from a total of 198 patients were studied,including 116 adnexae removed for non-neoplastic indications,53 serous cystadenomas,44 serous borderline tumors,and 41 low-grade serous carcinomas,which were collected from Qilu Hospital of Shandong University and University of Arizona in USA.Immunohistochemical single staining was used to detect the expressions of PAX8,tubulin,calretinin,and Ki-67 in the two groups,while immunohistochemical double staining of PAX8/calretinin was used to figure out the immunophenotype of various ovarian epithelial inclusions in a more intuitive way.Results With immunohistochemical single staining of PAX8 and calretinin,the vast majority (90％,54/60) of ovarian surface epithelia displayed a mesothelial phenotype [calretinin(+),PAX8 (-)],whereas 10％ (6/60)of the cases displayed foci with tubal phenotype [calretinin(-),PAX8 (+)].In contrast,most (79％,728/921 ) of the ovarian epithelial inclusions displayed a tubal phenotype,though 21％ (193/921) of the ovarian epithelial inclusions showed a mesothelial phenotype.It was further proved by immunohistochemical double staining of PAX8/ calretinin.Secretory and ciliated cells were found in the ovarian epithelial inclusions with tubal phenotype.There was a progressive increase in the secretory/ciliated cells ratio and proliferative index,from ovarian epithelial inclusions/cystadenomas to borderline tumors to low-grade serous carcinoma,according to the expression of tubulin and Ki-67.Conclusions The findings make a strong argument that the ovarian epithelial inclusions displaying a tubal phenotype with PAX8 (+),calretinin (-) is likely derived from fallopian tube rather than through Mullerian metaplasia from ovarian surface epithelium.The increasing trend of secretory/ciliated cells ratio and proliferative index from ovarian epithelial inclusions/cystadenomas to borderline tumors to low-grade serous carcinoma indicates that the latter is a clonal expansion of secretory cells.Genetic and molecular studies are needed to further confirm these findings.