Proliferation of Submesothelial Mesenchymal Cells during Early Phase of Serosal Thickening in the Rabbit Bladder Is Accompanied by Transient Keratin 18 Expression

Partial outlet obstruction of the rabbit bladder induces serosal thickening and smooth muscle (SM) hypertrophy. Within thickened serosa, submesothelial (mesenchymal) cells differentiate into SM cells after 30 days of obstruction [S. Buoroet al. Lab. Invest.69, 589–602, 1993]. Here, we show that submesothelial cells transiently express keratin (K) 18 but not K8 soon after obstruction. We investigated a possible relationship between keratin expression and cell proliferation/differentiationin vivoandin vitro.The results of this study indicate that expression of K18 is spatiotemporally related to the pattern of cell proliferation with respect to the localization of an elastic membrane which divides the thickened serosa into an “extrinsic” and an “intrinsic” region. Moreover, K18 is not present in bladder mesenchyma during early development, indicating that its expression in the adult is not attributable to a dedifferentiation process. However, simultaneous K18, K8, and desmoplakin (DP) expression can be induced in normal and thickened serosa upon treatment with bromo-deoxyuridine. Our results indicate that K18 is a marker of proliferating mesenchymal cells in rabbit serosa, whereas the combined expression of K18, K8, and DP might be related to the hypothesized alterations in the stability of gene expression. A model is proposed in which keratin-containing submesothelial cells can act as a “transit” cell phenotype involved in both regenerating mesothelial cells and formation of SM cells.     

QuantiFERON-TB GOLD ELISA assay for the detection of Mycobacterium tuberculosis-specific antigens in blood specimens of HIV-positive patients in a high-burden country

Tuberculosis is a life-threatening infection worldwide. Despite improvements in therapy, it results in 2 million deaths and 9 million new cases annually. This study evaluated the use of the QuantiFERON-TB GOLD enzyme-linked immunosorbent assay in a high HIV/TB burden setting in an ARV clinic at the Tshwane District Hospital, South Africa. The sensitivity and specificity of the QF assay in the clinic were 30% (9/30) and 63% (19/30), respectively, when compared with the gold standard culture results. Analysis also suggested that the sensitivity of the QuantiFERON assay is determined by a limiting patient CD4 value between 150 and 200.     

Detection of blaSHV, blaTEM and blaCTX-M antibiotic resistance genes in randomly selected bacterial pathogens from the Steve Biko Academic Hospital

Extended-spectrum β-lactamases (ESBLs) are considered to be one of the most important antibiotic resistance mechanisms. This study reported the ESBL-producing genes in 53 randomly selected clinical bacterial isolates from the Steve Biko Academic Hospital. The presence of the bla _SHV, bla _TEM and bla _CTX-M genes was determined, and the overall prevalence of these genes detected in this study was 87% (46/53) in comparison with the literature; these results were higher when compared with 33% for Escherichia coli in Europe and 0.8% in Denmark for similar pathogens. These research findings indicated that it is crucial to routinely monitor the prevalence of these resistance genes.     

Towards precision medicine in diabetes? A critical review of glucotypes

In response to a study previously published in PLOS Biology, this Formal Comment thoroughly examines the concept of ’glucotypes’ with regard to its generalisability, interpretability and relationship to more traditional measures used to describe data from continuous glucose monitoring.

Although the promise of precision medicine has led to advances in the recognition and treatment of rare monogenic forms of diabetes, its impact on prevention and treatment of more common forms of diabetes has been underwhelming [1]. Several approaches to the subclassification of individuals with, or at high risk of, type 2 diabetes have been published recently [2–4]. Hall and colleagues introduced the concept of “glucotypes” in a research article [3] that has received enormous attention in the highest impact scientific journals [5–8], mostly in relation to precision medicine. The authors developed an algorithm to identify patterns of glucose fluctuations based on continuous glucose monitoring (CGM). They named the 3 identified patterns: “low variability,” “moderate variability,” and “severe variability” glucotypes. Each individual was characterised by the proportion of time spent in the 3 glucotypes and was assigned to an overall glucotype based on the highest proportion. They argued that glucotypes provide the advantage of taking into account a more detailed picture of glucose dynamics, in contrast to commonly used single time point or average-based measures, revealing subphenotypes within traditional diagnostic categories of glucose regulation. Even though the study was based on data from only 57 individuals without a prior diabetes diagnosis, others have interpreted the results as indicating that glucotypes might identify individuals at an early stage of glucose dysregulation, suggesting a potential role in diabetes risk stratification and prevention [5]. However, before glucotypes can become “an important tool in early identification of those at risk for type 2 diabetes” [3], the concept requires thorough validation. Therefore, we explore the generalisability and interpretability of glucotypes and their relationship to traditional CGM-based measures.We used data from The Maastricht Study [9] and the PRE-D Trial [10] comprising a total number of 770 diabetes-free individuals with a 7-day CGM registration. We observed that the average proportion of time spent in the low variability glucotype was low both in The Maastricht Study (6%) and the PRE-D Trial (4%), compared to 20% in the original study. A reason for the difference may be that our study populations were on average 11 to 12 years older and that the PRE-D Trial (n = 116) included only overweight and obese individuals with prediabetes. In The Maastricht Study, the median (Q1 to Q3) body mass index was 25.9 kg/m² (23.4 to 28.7), and 72% had normal glucose tolerance. As a logical consequence, the severe glucotype was most common in the PRE-D Trial (55%). Regardless, our data show that the initial estimates of the different glucotype prevalences do not necessarily generalise to other populations, especially in age groups at increased risk of type 2 diabetes.Hall and colleagues described glucotypes as a new measure of glucose variability, a clinically relevant metric of glycaemic patterns [3]. In the figures accompanying the original publication, the low variability pattern was characterised by both the lowest mean glucose level and variation, while the severe pattern had both the highest mean glucose level and variation. As such, these examples did not give an intuition whether glucotypes were predominantly driven by glucose variability or by mean glucose levels. We therefore present 3 examples from the PRE-D Trial (Fig 1). The first 2 profiles are very similar with regard to glucose variability. Thus, the driver of the most severe glucotype of the second participant is clearly the slightly higher mean glycaemic level. Also, even though the third participant has a much larger variation than the first two, the proportion of time in the severe glucotype is not higher than for the second participant as one would expect from a classical measure of glucose variability. To investigate this further, we assessed the association between glucotypes and classical CGM measures, i.e., the mean CGM glucose level (Fig 2A) and the coefficient of variation (Fig 2B) in The Maastricht Study. The scatterplots show a clear association between the mean CGM glucose and glucotypes. They also suggest that participants with a high proportion of time in the moderate glucotype do not have high variation in glucose. Rather than a biological feature, this may well be a methodological consequence of being assigned to the middle cluster. If large fluctuations were present, glucose levels would reach either low or high values, resulting in a higher proportion of time spent in the low or severe glucotypes, respectively (assuming a strong association between glucotypes and mean CGM glucose). Therefore, we decided to quantify this association using regression analysis where glucotype proportions were the outcomes, and the mean CGM glucose concentration was the independent variable modelled with natural cubic splines (more details on the specification of the models are given in Supporting information S1–S3 Codes). Then, we used the equation estimated in The Maastricht Study to predict glucotypes in the external validation sample (PRE-D Trial, Fig 2C). First, similarly to Hall and colleagues, we assigned individuals to the pattern with the highest proportion of time and then compared the predicted and the observed glucotypes. We found that in 107 out of 116 individuals, the glucotype was predicted correctly when using only the mean CGM glucose value. When considering the glucotypes as continuous proportions of time, the root mean squared errors (RMSEs) were 0.05, 0.09, and 0.07 for the low, moderate, and severe variability glucotypes, respectively, indicating good predictive ability. These results demonstrate that glucotypes either mainly reflect the mean CGM glucose level or do not translate to external datasets (e.g., due to overfitting). To investigate this further, we conducted the same analyses as described for the PRE-D Trial in the original data from Hall and colleagues and found a slightly weaker, but still strong association between mean CGM glucose levels and glucotypes. Using the regression model from The Maastricht Study, we could correctly predict 79% of the glucotypes, while the RMSEs were 0.11, 0.15, and 0.13.Open in a separate windowFig 1Example CGM profiles of participants in the PRE-D Trial with corresponding proportion of time spent in different glucotypes and conventional measures (mean and CV).CGM, continuous glucose monitoring; CV, coefficient of variation.Open in a separate windowFig 2Observed proportion of time spent in the 3 glucotypes by mean CGM glucose (A) and coefficient of variation (B) in The Maastricht Study, and by mean CGM glucose in the PRE-D Trial (C) alongside predicted proportions based on the regression analysis in The Maastricht Study. CGM, continuous glucose monitoring.Although the transformation of continuous measures into categorical ones is a common procedure in clinical research, assigning individuals to the glucotype with the highest proportion of time runs very much against the “precision” tenet of precision medicine. In line with this, a recent study has demonstrated how simple clinical features outperformed clusters in predicting relevant clinical outcomes [11]. This is especially problematic when a method does not provide clear separation between clusters, which can be quantified by calculating relative entropy [12]. A relative entropy of zero would mean that all individuals spend one-third of the time in each of the 3 glucotypes, while a value of one would indicate that each individual spends the entire time period in only one of the 3 glucotypes. In the original cohort of Hall and colleagues [3], we calculated a relative entropy of 0.24 indicating that cluster separation is far from optimal and together with the previous results question the claim that the glucotype is really a “more comprehensive measure of the pattern of glucose excursions than the standard laboratory tests in current use” [3].In conclusion, we demonstrate in 2 large, external datasets, that the assessment of glucotypes does not offer more novel insights than the mean CGM glucose, highlighting the importance of large development datasets and external validation for data-driven algorithms. As CGM is becoming more widely used in large clinical studies also among individuals without diabetes, glucose patterns derived from CGMs will be an important focus area in future diabetes research. However, it is important that scientific scrutiny precedes the introduction of emerging tools with a promise of identifying individuals at high risk of type 2 diabetes and its late complications at an earlier stage of disease progression, especially in an observational setting. Furthermore, future efforts towards precision medicine for diabetes prevention and treatment should go beyond the glucocentric approach we have seen so far. We know that hyperglycaemia is a late feature of diabetes development and that patients benefit most from a multifactorial treatment approach [13]. A multifactorial approach, with relevance to the aetiology of micro- and macrovascular complications, may also yield a more clinically useful risk stratification of nondiabetic individuals [14]. Even so, if we aim for precision medicine, we should aim to retain as much precision as possible at every step of the process, by treating determinants and outcomes as continuous measures if possible and by retaining information on the uncertainty of any hard classification such as cluster membership.     

The primary structure of a feline class I gene: Striking similarity toHLA-A

The sequence of a feline class I pseudogene and its comparison with class I genes from other species is presented. The gene isolated is a pseudogene because of the presence of four stop codons and two frame shift mutations in the first-and second-domain encoding exons, as well as a mutation in a splice acceptor site in the third intron. By sequence comparison with the other class I sequences determined to date, theFLA pseudogene is most closely related to theHLA-A locus products (88% nucleotide identity). The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M27192.