Tratamiento con rituximab para las miopatías necrosantes autoinmunes asociadas al anti-SRP: caso clínico y revisión de la evidencia / Rituximab therapy in necrotizing autoimmune myopathy associated with anti-SRP antibody: A clinical case review

Las miopatías necrosantes autoinmunes (NAM) son unas entidades raras y emergentes de las miopatías inflamatorias idiopáticas (MII). Su presencia se ha asociado a conectivopatías, infecciones virales, neoplasias, anticuerpos frente a la partícula de reconocimiento de la señal (anti-SRP) y a anticuerpos frente a la proteína 3-hidroxi-3-metilglutaril-coenzima A reductasa (HMG-CR) (asociada al empleo o no de estatinas). Las NAM asociadas al anti-SRP presentan unas características clínicas y anatomopatológicas que las diferencian del resto de las MII, resultando en un peor pronóstico. Hasta el momento han sido muy pocos los casos publicados tratados con rituximab y con diferente respuesta clínica. Presentamos el caso de una mujer de 30 años con NAM asociada al anti-SRP refractaria a tratamiento convencional inmunosupresor y que ha sido controlada a largo plazo con la combinación de rituximab, metotrexato y corticoides

Necrotizing autoimmune myopathy (NAM) is a rare and emerging entity of idiopathic inflammatory myopathy (IIM). They have been associated with connective tissue disorders, viral infections, malignancy, anti-signal recognition particle (SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase protein (with or without the use of statins). Anti-SRP associated NAM has different clinical and histological characteristics that differentiate them from other IIM, resulting in a poor prognosis. Very few cases treated with rituximab have been published, with varying clinical response. Here we describe a case of anti-SRP associated NAM refractory to conventional immunosuppressants and its successful long-term management with the combination of rituximab, corticosteroids and methotrexate

SRP, FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk.

Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs. In this study, we identify DjlC and Flk as bona fide Escherichia coli TAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.

Clinical and parasitological factors in parasite persistence after treatment and clinical cure of cutaneous leishmaniasis.

BACKGROUND: The determinants of parasite persistence or elimination after treatment and clinical resolution of cutaneous leishmaniasis (CL) are unknown. We investigated clinical and parasitological parameters associated with the presence and viability of Leishmania after treatment and resolution of CL caused by L. Viannia. METHODS: Seventy patients who were treated with meglumine antimoniate (n = 38) or miltefosine (n = 32) and cured, were included in this study. Leishmania persistence and viability were determined by detection of kDNA and 7SLRNA transcripts, respectively, before, at the end of treatment (EoT), and 13 weeks after initiation of treatment in lesions and swabs of nasal and tonsillar mucosa. RESULTS: Sixty percent of patients (42/70) had evidence of Leishmania persistence at EoT and 30% (9/30) 13 weeks after treatment initiation. A previous episode of CL was found to be a protective factor for detectable Leishmania persistence (OR: 0.16, 95%CI: 0.03-0.92). kDNA genotyping could not discern differences between parasite populations that persisted and those isolated at diagnosis. CONCLUSIONS: Leishmania persist in skin and mucosal tissues in a high proportion of patients who achieved therapeutic cure of CL. This finding prompts assessment of the contribution of persistent infection in transmission and endemicity of CL, and in disease reactivation and protective immunity.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

BACKGROUND: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. METHODS: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. PRINCIPAL FINDINGS: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. CONCLUSIONS: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

Natural infection of North African gundi (Ctenodactylus gundi) by Leishmania tropica in the focus of cutaneous leishmaniasis, Southeast Tunisia.

North African gundis (Ctenodactylus gundi) were trapped in the Leishmania (L.) tropica focus of cutaneous leishmaniasis, situated in southeast Tunisia and evaluated for Leishmania infection by real-time kinetoplast DNA polymerase chain reaction (PCR). Species identification was performed by internal transcribed spacer one (ITS1)-PCR-restriction fragment length polymorphism (RFLP) and high-resolution melting (HRM) analysis of the 7SL RNA gene. Real-time PCR on blood was positive in 6 of 13 (46.2%) tested gundis. Leishmania tropica was identified in five infected gundis and Leishmania major in one specimen. Alignments of the ITS-1 DNA sequences and 7S-HRM curves analysis indicated that similar genotypes were present in humans, a sandfly, and gundis from the same region suggesting a potential role of this rodent as reservoir host of L. tropica in southeast Tunisia.

Prevalence of canine leishmaniasis in Beichuan County, Sichuan, China and phylogenetic evidence for an undescribed Leishmania sp. in China based on 7SL RNA.

BACKGROUND: Leishmaniasis is a vector-borne disease, which is still endemic in the west and northwest area of China. Canines are the major reservoirs of Leishmania, the etiological agent of human visceral leishmaniasis. Phlebotomus chinensis is the main transmission vector of zoonotic visceral leishmaniasis (ZVL). METHODS: In this study, rK39 dip-stick, ELISA and PCR methods were used to investigate the prevalence of canine leishmaniasis (CanL) in Beichuan County, Sichuan Province, China. RESULTS: Among the 86 dogs which were included in the study, 13 dogs were positive using the dip-stick test (15.12%), while 8 dogs were positive using ELISA (9.30%) and 19 dogs were positive for PCR (22.03%). In total, 32 dogs were positive for one or more tests (37.21%). Interestingly, phylogenetic analysis based on the partial 7SL RNA fragment provided evidence that an undescribed Leishmania species, which is clearly a causative agent of CanL and human visceral leishmaniasis, does exist in China. This result is consistent with our previous study. CONCLUSIONS: Our work confirmed that canine leishmaniasis is still prevalent in Beichuan County. Further control is urgently needed, as canine leishmaniasis is of great public health importance. The phylogenetic analysis based on 7SL RNA segment provides evidence for the existence of an undescribed Leishmania sp. in China.

7SL RNA is retained in HIV-1 minimal virus-like particles as an S-domain fragment.

HIV-1 is known to package several small cellular RNAs in addition to its genome. Previous work consistently demonstrated that the host structural RNA 7SL is abundant in HIV-1 virions but has yielded conflicting results regarding whether 7SL is present in minimal, assembly-competent virus-like particles (VLPs). Here, we demonstrate that minimal HIV-1 VLPs retain 7SL RNA primarily as an endoribonucleolytic fragment, referred to as 7SL remnant (7SLrem). Nuclease mapping showed that 7SLrem is a 111-nucleotide internal portion of 7SL, with 5' and 3' ends corresponding to unpaired loops in the 7SL two-dimensional structure. Analysis of VLPs comprised of different subsets of Gag domains revealed that all NC-positive VLPs contained intact 7SL while the presence of 7SLrem correlated with the absence of the NC domain. Because 7SLrem, which maps to the 7SL S domain, was not detectable in infected cells, we propose a model whereby the species recruited to assembling VLPs is intact 7SL RNA, with 7SLrem produced by an endoribonuclease in the absence of NC. Since recruitment of 7SL RNA was a conserved feature of all tested minimal VLPs, our model further suggests that 7SL's recruitment is mediated, either directly or indirectly, through interactions with conserved features of all tested VLPs, such as the C-terminal domain of CA.

Alteraciones histológicas y moleculares en miopatías inflamatorias / Histological and molecular alterations in inflammatory myopathies

Los hallazgos de las biopsias musculares en las miopatías inflamatorias idiopáticas (MII) son divididos en: a) infiltrados inflamatorios endomisiales compuestos por células T CD8+, CD4+ y macrófagos (MΦ), y b) infiltrados perivasculares compuestos por células T CD4+, MΦ y células B. El infiltrado endomisial sugiere una reacción inmune directa hacia las fibras musculares y fue sugerido como típico para la polimiositis (PM) y la miositis de cuerpos de inclusión, mientras que el infiltrado perivascular indica una reacción inmunitaria contra los vasos sanguíneos típicos en dermatomiositis. Se ha demostrado que autoantígenos relacionados con MII (Mi-2, Jo-1, OJ, PL12, Ku, PM/Scl) son específicamente segmentados por la granzima B durante la apoptosis inducida por linfocitos T citotóxicos. En este capítulo abordamos las alteraciones histológicas y moleculares así como un grupo de anticuerpos no estudiados en la población mexicana, como son los anticuerpos anti-SRP (signal recognition particle ‘partícula de reconocimiento de señal’), en especial autoanticuerpos anti-SRP54 y anti-SRP72 que presentan clínicamente una enfermedad aguda e involucramiento cardíaco severo, de pronóstico severo y pobres respuestas a las terapias convencionales (AU)

The histological findings in muscle biopsies of inflammatory myopathies have been divided into 2 groups: A) Endomisial infiltrates mainly by T CD8+, CD4+ and macrophages and B) Perivascular infiltrates by CD4+, B cells and macrophages. The first kind of infiltrate suggests an immune reaction against muscle fibers very common in PM and inclusion body myositis, On the other hand the perivascular infiltrate is a hallmark of DM. It has been shown that autoantigens related with myopathies such as Mi-2, Jo-1, OJ, PL12, Ku, PM/Scl are able to suffer proteolytic cleavage by granzyme B and other stimulus induced by cytotoxic T cells. In this chapter we will review the histological and molecular findings of inflammatory myopathies but we will also discuss a special group of myopathies related to the presence of antibodies against the SRP complex, in particular the SRP72 and SRP54 antibodies, which are associated with a poor prognosis and clinical outcome and present an inadequate response to conventional treatment (AU)

Characterization of the Yarrowia lipolytica YlSRP72 gene, a component of the yeast signal recognition particle

The Yarrowia lipolytica SRP72 gene product (YlSRP72), a homolog of the 72-kDa subunit of the mammalian SRP, encodes a putative protein of 602 amino acids. Northern blot analysis revealed a unique YlSRP72-specific transcript of 1.8 kb. The deduced amino acid sequence showed higher identities with the Srp72 proteins of euascomycetes than with hemiascomycetes. Chromosomal hybridization experiments showed that the YlSRP72 gene is located in chromosome V of the standard E150 strain of Y. lipolytica. Fluorescent microscopy revealed that the YlSRP72-GFP fusion protein was expressed in the cytoplasm and nucleus. The YlSRP72 gene was interrupted by the pop-out method; however, deletion of the gene proved to be lethal. This is in contrast to the results described for the Saccharomyces cerevisiae SRP72 gene, which is not essential for cell growth, and supports our previous finding with another component of the yeast recognition particle, YlSEC65. The present work suggests that SRP-dependent targeting is the main secretory pathway in Y. lipolytica, as has been described for higher eukaryotes (AU)

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7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles.

The virion incorporation of 7SL, the RNA component of the host signal recognition particle (SRP), has been shown for several simple retroviruses. Data here demonstrate that 7SL is also packaged by HIV-1, in sevenfold molar excess of genomic RNA. Viral determinants of HIV-1 genome and primer tRNA packaging were not required for 7SL incorporation, as virus-like particles with only minimal assembly components efficiently packaged 7SL. The majority of 7SL within cells resides in ribonucleoprotein complexes bound by SRP proteins, and most SRP protein exists in signal recognition particles. However, Western blot comparison of virion and cell samples revealed that there is at least 25-fold less SRP p54 protein per 7SL RNA in HIV-1 particles than in cells. Comparing 7SL:actin mRNA ratios in virions and cells revealed that 7SL RNA appears selectively enriched in virions.

Nonrandom packaging of host RNAs in moloney murine leukemia virus.

Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA(Met) were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Psi- mRNAs and some other host RNAs may be specifically excluded from assembly sites.

Novel autoantibodies against 7SL RNA in patients with polymyositis/dermatomyositis.

OBJECTIVE: Autoantibodies against signal recognition particle (SRP) are detected in patients with polymyositis/dermatomyositis (PM/DM). The SRP consists of 7SL RNA and 6 protein components. We examined autoantibodies against deproteinized 7SL RNA in PM/DM patients with anti-SRP antibodies and evaluated the association of anti-7SL RNA antibodies with PM/DM clinically and serologically. METHODS: Sera from 10 Japanese and 22 North American PM/DM patients with anti-SRP antibodies were tested for the presence of anti-7SL RNA antibodies, using the sera to immunoprecipitate deproteinized RNA extracts derived from HeLa cells. RESULTS: The immunoprecipitation analysis indicated that 5 Japanese (50%) and one North American (5%) patient with anti-SRP antibodies had novel autoantibodies against deproteinized 7SL RNA. The frequency of anti-7SL RNA antibodies was significantly higher in Japanese than North American patients (p = 0.006). The presence of anti-7SL RNA antibodies appeared to be associated with DM (2 patients) and finger swelling (2 PM patients). The seasonal onset of the disease was different (p = 0.008) for Japanese PM/DM patients with anti-7SL RNA antibodies, who developed the disease between October and January (mean month November; p = 0.01) from that of patients without these antibodies, who developed it between June and August (mean month July; p = 0.01). CONCLUSION: Novel autoantibodies against 7SL RNA were identified in patients with PM/DM, and the presence of these antibodies was correlated to ethnic background, clinical features, and season of disease onset. These findings indicated that autoantibodies against 7SL RNA are a novel serological marker for a subset of PM/DM cases.

Signal recognition particle protein 19 is imported into the nucleus by importin 8 (RanBP8) and transportin.

The signal recognition particle (SRP) is a cytoplasmic RNA-protein complex that targets proteins to the rough endoplasmic reticulum. Although SRP functions in the cytoplasm, RNA microinjection and cDNA transfection experiments in animal cells, as well as genetic analyses in yeast, have indicated that SRP assembles in the nucleus. Nonetheless, the mechanisms responsible for nuclear-cytoplasmic transport of SRP RNA and SRP proteins are largely unknown. Here we show that the 19 kDa protein subunit of mammalian SRP, SRP19, was efficiently imported into the nucleus in vitro by two members of the importin beta superfamily of transport receptors, importin 8 and transportin; SRP19 was also imported less efficiently by several other members of the importin beta family. Although transportin is known to import a variety of proteins, SRP19 import is the first function assigned to importin 8. Furthermore, we show that a significant pool of endogenous SRP19 is located in the nucleus, as well as the nucleolus. Our results show that at least one mammalian SRP protein is specifically imported into the nucleus, by members of the importin beta family of transport receptors, and the findings add additional evidence for nuclear assembly of SRP.

Localization of signal recognition particle RNA in the nucleolus of mammalian cells.

The signal recognition particle (SRP) of eukaryotic cells is a cytoplasmic ribonucleoprotein machine that arrests the translational elongation of nascent secretory and membrane proteins and facilitates their transport into the endoplasmic reticulum. The spatial pathway of SRP RNA processing and ribonucleoprotein assembly in the cell is not known. In the present investigation, microinjection of fluorescently tagged SRP RNA into the nucleus of mammalian cells was used to examine its intranuclear sites of localization. Microinjection of SRP RNA into the nuclei of normal rat kidney (NRK) epithelial cells maintained at 37 degreesC on the microscope stage resulted in a very rapid initial localization in nucleoli, followed by a progressive decline of nucleolar signal and an increase of fluorescence at discrete sites in the cytoplasm. Nuclear microinjection of a molecule corresponding to a major portion of the Alu domain of SRP RNA revealed a pattern of rapid nucleolar localization followed by cytoplasmic appearance of signal that was similar to the results obtained with full-length SRP RNA. In contrast, a molecule corresponding to the S domain of SRP RNA did not display nucleolar localization to the extent observed with full-length SRP RNA. An SRP RNA molecule lacking helix 6 of the S domain displayed normal nucleolar localization, whereas one lacking helix 8 of the S domain did not. These results, obtained by direct, real-time observation of fluorescent RNA molecules inside the nucleus of living mammalian cells, suggest that the processing of SRP RNA or its ribonucleoprotein assembly into the SRP involves a nucleolar phase.

Concentrations of 4.5S RNA and Ffh protein in Escherichia coli: the stability of Ffh protein is dependent on the concentration of 4.5S RNA.

We measured the concentrations of both 4.5S RNA and Ffh protein under a variety of growth conditions and found that there were 400 molecules of 4.5S RNA per 10,000 ribosomes in wild-type cells and that the concentration of Ffh protein was one-fourth of that. This difference in concentration is 1 order of magnitude less than that previously reported but still significant. Pulse-chase labeling experiments indicated that Ffh protein is unstable in cells carrying ffh on high-copy-number plasmids and that simultaneous overproduction of 4.5S RNA stabilizes Ffh protein. Our analyses show that free Ffh protein is degraded with a half-life of approximately 20 min. We also tested whether three previously isolated suppressors of 4.5S RNA deficiency could reduce the requirement for Ffh protein. Since the two sffE suppressors do not suppress the Ffh requirement, we suggest that 4.5S RNA either acts in a sequential reaction with Ffh or has two functions.