Background Emerging studies indicate that some COVID-19 patients suffer from persistent symptoms including breathlessness and chronic fatigue; however the long-term immune response in these patients presently remains ill-defined. Methods Here we describe the phenotypic and functional characteristics of B and T cells in hospitalised COVID-19 patients during acute disease and at 3-6 months of convalescence. Findings We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed continued alterations with persistence of a cytotoxic programme evident in CD8 + T cells as well as elevated production of type-1 cytokines and IL-17. Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/IL-10 cytokine imbalance in response to toll-like receptor activation, skewed towards a pro-inflammatory phenotype. Whereas the frequency of IL-6 + B cells was restored in convalescent patients irrespective of clinical outcome, recovery of IL-10 + B cells was associated with resolution of lung pathology. Conclusions Our data detail lymphocyte alterations in previously hospitalized COVID-19 patients up to 6 months following hospital discharge and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes, with one subgroup associated with poorer clinical outcome. We propose that alterations in B and T cell function following hospitalisation with COVID-19 could impact longer term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. Funding Provided by UKRI, Lister Institute of Preventative Medicine, The Wellcome Trust, The Kennedy Trust for Rheumatology Research and 3M Global Giving.

Type 2 diabetes (T2D) is a common, polygenic chronic disease with high heritability. The purpose of this whole-genome association study was to discover novel T2D-associated genes. We genotyped 500 familial cases and 497 controls with >300,000 HapMap-derived tagging single-nucleotide-polymorphism (SNP) markers. When a stringent statistical correction for multiple testing was used, the only significant SNP was at TCF7L2, which has already been discovered and confirmed as a T2D-susceptibility gene. For a replication study, we selected 10 SNPs in six chromosomal regions with the strongest association (singly or as part of a haplotype) for retesting in an independent case-control set including 2,573 T2D cases and 2,776 controls. The most significant replicated result was found at the AHI1-LOC441171 gene region.

Information on the extent to which xenobiotics interact with P-glycoprotein (PGP) during transit through the intestine is crucial in determining the influence of PGP on oral drug absorption. We have recently described a novel use of isolated ileum from PGP-deficient mdr1a(Ϫ/Ϫ) mice to resolve PGPand non-PGP-dependent drug efflux and provide a definitive measure of intrinsic drug permeability without recourse to inhibitors (Stephens et al., 2002). The present study uses this approach to investigate the impact of PGP on intestinal permeability of paclitaxel and digoxin in different regions of the mouse intestine (jejunum, ileum, and proximal and distal colon). Absorption of paclitaxel and digoxin in tissues from wild-type mice was low and showed little regional variation. In contrast, absorption of both drugs was markedly higher in mdr1a(Ϫ/Ϫ) intestine, although the increase was highly region-dependent, with the ileum and distal colon showing the greatest effect and much smaller changes in the jejunum and proximal colon. These effects were accompanied by the abolition of paclitaxel and digoxin secretion in mdr1a(Ϫ/Ϫ) mice, suggesting that regional variations in intestinal permeability are masked by differential PGP expression, confirmed by immunoblotting studies. Propranolol permeability, which is not influenced by PGP, showed similar regional variation in both wild-type and mdr1a(Ϫ/Ϫ) tissues, suggesting that differences are at the level of transcellular permeability. These data suggest that the ileum and the distal colon are regions of relatively high transcellular permeability for xenobiotics that are compensated by enhanced expression of PGP.

The use of ancient DNA in paleopathological studies of tuberculosis has largely been restricted to confirmation of disease identifications made by skeletal analysis; few attempts at obtaining genotype data from archaeological samples have been made because of the need to perform different PCRs for each genetic locus being studied in an ancient DNA extract. We used a next generation sequencing approach involving hybridization capture directed at specific polymorphic regions of the Mycobacterium tuberculosis genome to identify a detailed genotype for a historic strain of M. tuberculosis from an individual buried in the 19th century St. George's Crypt, Leeds, West Yorkshire, England. We obtained 664,500 sequencing by oligonucleotide ligation and detection (SOLiD) reads that mapped to the targeted regions of the M. tuberculosis genome; the coverage included 218 of 247 SNPs, 10 of 11 insertion/deletion regions, and the repeat elements IS1081 and IS6110. The accuracy of the SOLiD data was checked by conventional PCRs directed at 11 SNPs and two insertion/deletions. The data placed the historic strain of M. tuberculosis in a group that is uncommon today, but it is known to have been present in North America in the early 20th century. Our results show the use of hybridization capture followed by next generation sequencing as a means of obtaining detailed genotypes of ancient varieties of M. tuberculosis, potentially enabling meaningful comparisons between strains from different geographic locations and different periods in the past. biomolecular archaeology | paleopathology

1 Intestinal xenobiotic transporters are a signi®cant barrier to the absorption of many orally administered drugs. P-glycoprotein (PGP) is the best known, but several others, including members of the multidrug resistance-associated protein (MRP) family, are also expressed. De®nitive information on their precise e ect on intestinal drug permeability is scarce due to a lack of speci®c inhibitors and the di culty of studying non-PGP activity in the presence of high PGP expression. 2 We have investigated the in vitro use of intestinal tissues from PGP knockout (mdr1a (7/7)) mice as a tool for dissecting the mechanisms of intestinal drug e ux. The permeability characteristics of digoxin (DIG), paclitaxel (TAX) and etoposide (ETOP) were measured in ileum from mdr1a (7/7) and wild-type (FVB) mice mounted in Ussing chambers. 3 DIG and TAX exhibited marked e ux across FVB tissues (B-A : A-B apparent permeability (P app ) ratio 10 and 17 respectively) which was absent in mdr1a (7/7) tissues, con®rming that PGP is the sole route of intestinal e ux for these compounds. The A-B P app of both compounds was 3 ± 5 fold higher in mdr1a (7/7) than in FVB. 4 Polarized transport of ETOP in FVB tissues was reduced but not abolished in mdr1a (7/7) tissues. Residual ETOP e ux in mdr1a (7/7) tissues was abolished by the MRP inhibitor MK571, indicating involvement of both PGP and MRP. 5 MK571 abolished calcein e ux in mdr1a (7/7) tissues, while quinidine had no parallel e ect in FVB tissues, suggesting involvement of MRP but not PGP. 6 Tissues from mdr1a (7/7) mice provide a novel approach for investigating the in¯uence of PGP ablation on intestinal permeability and for resolving PGP and non-PGP mechanisms that modulate drug permeability.

Insulin and insulin-like growth factor-1 (IGF-1) act through highly homologous receptors that engage similar intracellular signaling pathways, yet these hormones serve largely distinct physiological roles in the control of metabolism and growth, respectively. In an attempt to uncover the molecular mechanisms underlying their divergent functions, we compared insulin receptor (IR) and IGF-1 receptor (IGF-1R) regulation of gene expression by microarray analysis, using 3T3-L1 cells expressing either TrkC/IR or TrkC/IGF-1R chimeric receptors to ensure the highly selective activation of each receptor tyrosine kinase. Following stimulation of the chimeric receptors for 4 h, we detected 11 genes to be differentially regulated, of which 10 were up-regulated to a greater extent by the IGF-1R. These included genes involved in adhesion, transcription, transport, and proliferation. The expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen, was markedly increased by IGF-1R but not IR activation. This effect was dependent on MAPK, but not phosphatidylinositol 3-kinase, and did not require an autocrine loop through the epidermal growth factor receptor. HB-EGF mitogenic activity was detectable in the medium of 3T3-L1 preadipocytes expressing activated IGF-1R but not IR, indicating that the transcriptional response is accompanied by a parallel increase in mature HB-EGF protein. The differential abilities of the IR and IGF-1R tyrosine kinases to stimulate the synthesis and release of a growth factor may provide, at least in part, an explanation for the greater role of the IGF-1R in the control of cellular proliferation.

The dysregulation of the IGF system has been implicated in the pathogenesis of obesity, diabetes, and diabetes complications such as nephropathy, but little is known about the genomics of the IGF system in health and disease. We genotyped 13 single nucleotide polymorphisms (SNPs) in IGFBP1 gene in 732 representative type 2 diabetic patients from the Salford Diabetes Register. Of the 13 SNPs, 8 were polymorphic and 7 of those had minor allele frequencies >0.1, one of which was in the gene promoter and one of which was nonsynonymous in exon 4. The minor alleles of these SNPs and two others were associated with a reduced prevalence of diabetic nephropathy. Haplotype analysis revealed that 97% of the genetic variation for IGFBP1 in the population sample could be accounted for using two of the "reno-protective" SNPs, with other SNPs adding little extra information. One of these two SNPs was the nonsynonymous mutation in exon 4, lying close to the integrinbinding RGD motif, which is thought to affect tissue delivery of IGF-I by IGF-binding protein 1 (IGFBP-1), possibly suggesting a "reno-protective" effect via altered IGFBP-1 binding. In conclusion, we have described the first genomic markers to be associated with diabetic microvascular complications within the human IGFBP1 gene. Diabetes 54: [3547][3548][3549][3550][3551][3552][3553] 2005 T he IGF system is increasingly implicated in the development of type 2 diabetes and its complications. IGF-I and -II possess significant structural homology with insulin and consequently exert acute metabolic effects on carbohydrate and protein metabolism in addition to their potent mitogenic effects. IGF-I and -II have been linked to the pathogenesis of diabetic nephropathy, retinopathy, cardiovascular disease, deteriorating glucose tolerance, and weight gain (1-13).Unlike insulin, the effects of IGFs are modulated by specific IGF-binding proteins (IGFBPs), six of which have been well characterized (14,15). Of the six IGFBPs, IGFBP-1 is considered to be the principal acute regulator of IGF-I activity (16), forming a link between dietary ingestion, glucose metabolism, and the IGF axis. IGFBP-1 synthesis is limited to the liver, decidua, and kidney and is acutely inhibited by insulin via binding of hepatocyte nuclear factor-3␤ (HNF-3␤) to the insulin-response elements of the gene promoter. Conversely, increased IGFBP-1 production is primarily associated with cAMP and glucocorticoids acting via specific promoter elements, although increased synthesis is also associated with hyperglycemia (via upstream stimulatory factor 1 [USF-1]) and hypoxia (via the hypoxia-inducible transcription factor 1␣ [HIF-1␣] and nitric oxide) (17-22). Thus, IGFBP-1 is acutely metabolically regulated, unlike other IGFBPs (16). Rodent models and clinical observations have underlined the potential importance of IGFBP-1 dysregulation in the etiology of diabetic complications such as macrovascular disease and microvascular conditions such as nephropathy and retinopathy. Renal hypertrophy is an earlyonset feat...

Previous methods of estimating the volume of epithelial lining liquid (ELL) in the air-filled lung may be said to have suffered from some theoretical and practical deficiencies. To estimate the ELL volume (VELL) more accurately, a known amount of artificial lung liquid (LL) containing the impermeant tracer 125I-albumin was instilled intraluminally in 56 in situ perfused postnatal sheep lungs (aged 36 h to 12 wk), and the rate of change of LL volume was measured by changes in the tracer concentration. Linear regression of LL volume against time allowed calculation of the VELL at the time of instillation; it was found to be 0.37 +/- 0.15 ml/kg body wt, which is equivalent to a film of 0.15 +/- 0.06 micron mean depth. The median ELL protein concentration was 36.8 mg/ml ELL, i.e., 0.60 times the plasma concentration, which agrees with previously published estimates and which is similar to the interstitial protein concentration. We conclude that the VELL is very small and that there is unlikely to be a protein osmotic gradient across the pulmonary epithelium.