Clinical characteristics and outcomes of patients with inflammatory and autoimmune rheumatological diseases admitted for intensive care in Colombia.

OBJECTIVE: Contemporary studies reporting outcomes of critical care in patients with inflammatory and autoimmune rheumatological diseases are scarce. This study describes 15 years of experience from 2005-2019 in a Colombian referral hospital. METHODS: This observational, descriptive, consecutive case series study was performed on adult patients with inflammatory and autoimmune rheumatic diseases who were admitted to the intensive care unit (ICU) of the San Ignacio University Hospital in Bogotá (Colombia), from January 1, 2005, to December 21, 2019. We describe the sociodemographic characteristics, admission causes and criteria, lengths of stay, immunosuppressive treatment, systemic support, and mortality. RESULTS: The study included 300 patients with a median age of 48 years [interquartile range (IQR) 31-62 years], predominantly female (76%). Disease exacerbations (30%), infections (17.6%), and cardiovascular diseases (15%) were the main causes of admission. Respiratory failure (23%) most commonly caused by septic shock (24%) was the principal indication for intensive care admission. The most frequent infections were community-acquired pneumonia (11.6%) and soft-tissue infections (9%). In 40.3% of patients, inotropic and vasopressor support was required. The median length of stay was 4 days (IQR 2-8), and global mortality was 21.6%. CONCLUSIONS: Rheumatic diseases in the ICU are still associated with high morbidity and mortality. Patients with inflammatory and autoimmune rheumatic diseases require a meticulous clinical approach, strict clinical monitoring, frequent assessment of complications, evaluation of systemic support needs, and specific management.

Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.

The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of beta-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.

Detection of common and private mutations in the COL6A1 gene of patients with Bethlem myopathy.

BACKGROUND: Dominant mutations in COL6A1, COL6A2, and COL6A3, the three genes encoding collagen type VI, a ubiquitous extracellular matrix protein, are associated with Bethlem myopathy (BM) and Ullrich scleroatonic muscular dystrophy. METHODS: The authors devised a method to screen the entire coding sequence of the three genes by reverse transcriptase-PCR amplification of total RNA from skin fibroblasts and direct sequencing of the resulting 25 overlapping cDNA fragments covering 107 exons. RESULTS: Four splicing and four missense mutations were identified in 16 patients with BM, six of which are novel mutations in COL6A1. Both common and private mutations are localized in the alpha1 (VI) chain between the regions corresponding to the 3' end of the NH2-globular domain and the 5' end of the triple helix, encoded by exons 3 through 14. CONCLUSIONS: The clustering of the mutations in a relatively narrow area of the three collagen type VI chains in patients with Bethlem myopathy (BM) suggests that mutations in different regions could result in different phenotypes or in no phenotype at all. Moreover, the detection of mutations in only 60% of the patients suggests the existence of at least another gene associated with BM. The authors propose the direct sequencing of COL6 cDNAs as the first mutation screening analysis in BM, given the high number of exon-skipping events.

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI.

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription-PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A --> G substitution at nucleotide -2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G --> A substitution at nucleotide -1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far-the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

Glucose 6-phosphate dehydrogenase expression is less prone to variegation when driven by its own promoter.

The ability to transfer permanently genes into mammalian cells makes retroviruses suitable vectors for the ultimate purpose of treating inherited genetic disease. However, expression of the retrovirally transferred genes is variable (position effect and expression variegation) because retroviruses are highly susceptible to the influence of the host genome sequences which flank the integration site. We have investigated this phenomenon with respect to the human housekeeping enzyme, glucose 6-phosphate dehydrogenase (hG6PD). We have constructed retroviral vectors in which the hG6PD cDNA is driven by either of two conventional retroviral promoters and enhancers from the Moloney Murine Leukemia Virus (MMLV) and the Myeloproliferative Sarcoma Virus (MPSV) long terminal repeats (LTR) or by the hG6PD own promoter replacing most of enhancer and promoter LTR (GRU5). We have compared the activity of retrovirally transferred hG6PD driven by these promoters after retroviral integration in bulk cultures and in individual clones of murine fibroblasts. The level of hG6PD expressed by the hG6PD promoter of GRU5-G6PD was significantly lower than that expressed by conventional retroviral vectors. However, analysis of the single copy clones showed less variation of expression with GRU5-G6PD (coefficient of variation, CV, 35.5%) than with conventional vectors (CV, 58.9%). Thus we have several vectors competent for reliable transfer and expression of hG6PD. The hG6PD promoter provides reproducible expression of hG6PD and limits the variability of expression. This decreased variability is important in order to help ensuring a consistent level of delivery of the needed gene product in future therapeutic protocols.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer.

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Microheterogeneity in the distribution of the 844ins68 in the cystathionine beta-synthase gene in Italy.

Cystathionine beta-synthase (CBS) is an important enzyme for methionine metabolism. A common 844ins68 insertion variant in the CBS gene has been described. This 68-bp duplication of the intron 7-exon 8 boundary within the CBS gene already has been reported to be associated in cis with the T833C mutation. Heterozygosity for CBS deficiency is considered an important cause of hyperhomocysteinemia that strongly relates to cardiovascular disease, as well as homozygosity for another common variant, the C677T mutation of 5,10-methylene tetrahydrofolate reductase. We analysed the prevalence of the 844ins68 variant in the CBS gene in 595 unrelated apparently healthy individuals from nine Italian regions and in 133 patients with coronary artery disease. Our data confirm that the T833C mutation cosegregates in cis with the 844ins68 in all carriers of the insertion. Furthermore, no statistical difference was found in the insertion variant allele frequency between controls and coronary artery disease patients. Our study indicates a microheterogeneity in the distribution of the 844ins68 in the Italian population.

World distribution of the T833C/844INS68 CBS in cis double mutation: a reliable anthropological marker.

Mild hyperhomocysteinemia is associated to mutations either in cystathionine beta-synthase (CBS) or in 5,10-methylenetetrahydrofolate reductase (MTHFR) genes. In 1995, Sebastio et al. characterized a 68 bp insertion in cis with the most common CBS mutation (T833C) detected in homocystinuric patients. Recently, this double mutation has been detected in Italian and North-American controls. Compared to a group of patients affected by coronary artery disease, North-American controls showed not statistically significant difference. Moreover, Italian controls displayed a microheterogeneity in the mutant allele frequency distribution depending on their geographical origin (North or South of Italy). Aim of our study was to evaluate the prevalence of the double in cis mutation in different populations. We studied 377 healthy subjects belonging to various human groups. Genomic DNA, extracted from peripheral blood samples, was amplified using specific primers; PCR fragments were digested with Bsr I restriction enzyme to detect the double mutation. Our data show a significant heterogeneity among the populations studied, therefore this mutation turned out to be a reliable anthropogenetic marker. The distribution of the double mutation will contribute, with other DNA polymorphisms, to evaluate the genetic admixture of mixed populations such as Afro-Americans.

The region 3' to Calpha1 gene of human IG heavy chain displays a polymorphic duplicated sequence and encodes an RNA associated with polysomes.

A highly spread polymorphism flanking the 3. Calpha1 human IG heavy chain gene was identified. This polymorphism allowed the detection of an internal duplication within the 3' flanking region of both Calpha1 and Calpha2. This region has a regulatory function with four enhancer structures also present at the 3' end of the human Calpha2 as well as in that of mouse and rat single Calpha genes. The 5682-bp sequence of clone lambdapl8 described here starts 3' of Calpha1 and presents three open reading frames; one of them contains part of the tandem repeats with the 20-bp consensus described previously that is expressed in a poly(A)+ RNA and found in three dbEST clones of the human tonsillar cDNA library. Here, we demonstrate that in the CLF1 B lymphoblastoid cell line, this transcript is associated with polysomes. We also discuss the possibility of the presence of a new regulatory gene that does not encode an immunoglobulin and maps in the human IG heavy chain gene cluster.

The binding sites for Xenopus laevis FIII/YY1 in the first exon of L1 and L14 ribosomal protein genes are dispensable for promoter expression.

The genes coding for the ribosomal proteins (rp genes) L14 and L1 in the toad Xenopus laevis are contacted in the first exon by the frog protein, FIII/YY1, homolog of the human zinc-finger protein YY1, acting as repressor, activator and initiator of transcription. To investigate the functional significance of FIII/YY1 in the context of the two rp genes, the L14 region at nucleotide positions -105 to +44, including all of the first exon was linked to the chloramphenicol acetyltransferase (CAT) reporter gene; constructs with wild-type and mutated sites for FIII/YY1 were injected into nuclei of stage V-VI oocytes and analyzed for CAT activity. The same procedure was followed for constructs made with L1 sequences at nucleotide positions -17 to +1567. Mutations in the sites for FIII/YY1 did not change reporter activity, nor did overexpression of FIII/YY1 in the oocytes prior to injection with L1 and L14 constructs. Since oocytes are non-dividing cells, transfections were made of Xenopus kidney cells in culture with the same constructs and the results obtained in oocytes confirmed.

Role of platelet glycoprotein PL(A1/A2) polymorphism in restenosis after percutaneous transluminal coronary angioplasty.

This study suggests a macroheterogeneity in prevalence of platelet glycoprotein PL(A1/A2) polymorphism in different ethnic populations. In patients undergoing percutaneous transluminal coronary angioplasty, this polymorphism does not represent an independent risk factor but seems to be implicated in restenosis after percutaneous transluminal coronary angioplasty.

Growth-dependent and growth-independent translation of messengers for heterogeneous nuclear ribonucleoproteins.

The hnRNP A1 transcript has a relatively short 5'- untranslated region (UTR) starting with a pyrimidine tract similar to that of mRNAs encoded by the TOP [terminal oligo(pyrimidine)] genes in vertebrates. Such genes code for ribosomal proteins and for other proteins directly or indirectly involved in the production and function of the translation apparatus. As expected from the role of the pyrimidine tract in the translational regulation of TOP mRNAs, the A1 mRNA is more efficiently loaded onto polysomes in growing than in resting cells. On the other hand, a less stringent regulation with respect to that of other TOP mRNAs is observed, partially due to the presence of multiple transcription start sites within the pyrimidine tract, where transcripts with shorter TOP sequences are less sensitive to regulation. Thus, from the point of view of structural features and translation behaviour the A1 mRNA can be included in the class of TOP genes, suggesting a possible role of A1 in translation. Interestingly, a TOP-like behaviour was observed for hnRNP I mRNA but not for hnRNP C1/C2 and A2/B1 mRNAs, indicating the existence of two classes of hnRNPs with different translational regulation.

Prevalence of factor V Leiden mutation in non-European populations.

A difference in the prevalence of venous thromboembolism (TE) in major human groups has been described and an uneven distribution of FV Leiden mutation over the world has recently been reported. We investigated FV Leiden mutation in 584 apparently healthy subjects mostly from populations different from those previously investigated: 170 Europeans (Spanish, Italians), 101 sub-saharan Africans (Fon, Bariba, Berba, Dendi), 115 Asians (Indonesians, Chinese, Tharus), 57 Amerindians (Cayapa), 84 Afroamericans (Rio Cayapa, Viche), and 57 Ethiopians (Amhara, Oromo). The mutation was detected in only 1/115 Asian (Tharu) and in 5/170 Europeans (4 Italians, 1 Spanish). These data confirm that in non-Europeans the prevalence of FV mutation is at least 7 times lower than in Europeans and provide indirect evidence of a low prevalence not only of the FV Leiden gene but also of other genes leading to more severe thrombophilia. Finally, findings from the literature together with those pertaining to this study clearly show a marked heterogeneity among Europeans.

Structure and expression of ribosomal protein genes in Xenopus laevis.

In Xenopus laevis, as well as in other vertebrates, ribosomal proteins (r-proteins) are coded by a class of genes that share some organizational and structural features. One of these, also common to genes coding for other proteins involved in the translation apparatus synthesis and function, is the presence within their introns of sequences coding for small nucleolar RNAs. Another feature is the presence of common structures, mainly in the regions surrounding the 5' ends, involved in their coregulated expression. This is attained at various regulatory levels: transcriptional, posttranscriptional, and translational. Particular attention is given here to regulation at the translational level, which has been studied during Xenopus oogenesis and embryogenesis and also during nutritional changes of Xenopus cultured cells. This regulation, which responds to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA (ribosomal protein mRNA) engaged on polysomes. A typical 5' untranslated region characterizing all vertebrate rp-mRNAs analyzed to date is responsible for this translational behaviour: it is always short and starts with an 8-12 nucleotide polypyrimidine tract. This region binds in vitro some proteins that can represent putative trans-acting factors for this translational regulation.