External validation of the Oldham composite Covid-19 associated mortality model (OCCAM), a prognostic model for death in patients hospitalised with Covid-19.

OBJECTIVE: External validation of the Oldham Composite Covid-19 associated Mortality Model (OCCAM), a prognostic model for Covid-19 mortality in hospitalised patients comprised of age, history of hypertension, current or previous malignancy, admission platelet count < 150 × 103/µL, admission CRP ≥ 100 µg/mL, acute kidney injury (AKI), and radiographic evidence of > 50% total lung field infiltrates. PATIENTS AND METHODS: Retrospective study assessing discrimination (c-statistic) and calibration of OCCAM for death in hospital or within 30 days of discharge. 300 adults admitted to six district general and teaching hospitals in North West England for treatment of Covid-19 between September 2020 and February 2021 were included. RESULTS: Two hundred and ninety-seven patients were included in the validation cohort analysis, with a mortality rate of 32.8%. The c-statistic was 0.794 (95% confidence interval 0.742-0.847) vs. 0.805 (95% confidence interval 0.766 - 0.844) in the development cohort. Visual inspection of calibration plots demonstrate excellent calibration across risk groups, with a calibration slope for the external validation cohort of 0.963. CONCLUSION: The OCCAM model is an effective prognostic tool that can be utilised at the time of initial patient assessment to aid decisions around admission and discharge, use of therapeutics, and shared decision-making with patients. Clinicians should remain aware of the need for ongoing validation of all Covid-19 prognostic models in light of changes in host immunity and emerging variants.

Rapid characterization of folding and binding interactions with thermolabile ligands by DSC.

Differential scanning calorimetry (DSC) is a powerful technique for measuring tight biomolecular interactions. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Using experimental data for two DNA aptamers that bind to the thermolabile ligand cocaine, we present a new global fitting analysis that yields the complete set of folding and binding parameters for the initial and final forms of the ligand from a pair of DSC experiments, while accounting for the thermal conversion. Furthermore, we show that the rate constant for thermolabile ligand conversion may be obtained with only one additional DSC dataset.

A randomized trial of long-chain polyunsaturated fatty acid supplementation in infants with phenylketonuria.

Forty-two infants (20 males, 22 females) with classical phenylketonuria (PKU) entered a prospective, double-blind, randomized study to investigate the effects on biochemical and physiological outcomes of a phenylalanine-free infant formula containing a fat blend supplemented with the long-chain polyunsaturated fatty acids (LC-PUFA), docosahexaenoic acid (DHA, C22:6 n-3), and arachidonic acid (AA, C20:4 n-6). Between entry and 20 weeks (entry and 1y) of age, median DHA levels in erythrocyte membrane phospholipids decreased by 15% (22%) in the LC-PUFA supplemented group (n=21) and by 61% (64%) in the control group (p<0.001; n=18). A dietary supply of LC-PUFA in infants with PKU prevents the decline in DHA levels associated with a diet supplying minimal sources of LC-PUFA. DHA status in turn, independent of diet, may influence the maturation of the visual system in infants with PKU.

Immunodominant B-cell epitope in the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis cross-reacting with glutathione S-transferase.

The TB1-5 76C monoclonal antibody raised against a synthetic 60-mer peptide in the N-terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131-138 in Mce1A, and to cross-react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross-react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme-linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14-mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442-7). The epitope is located on the surface of Mce1A, at the distal beta-strand-loop region in the beta-domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1-5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti-Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.

Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis.

In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al. Scand J Immunol 1999;50:510-8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione-S-transferase (GST) fusion proteins in recombinant Escherichia coli. Polyclonal antibodies against a predicted B-cell epitope of each of the Mce1 proteins of M. tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin. These antibodies reacted specifically with the corresponding fusion protein, except for GST-Mce1F. A mouse monoclonal antibody, TB1-5 76C, raised against a synthetic 60-mer peptide corresponding to the residues 106-165 in the N-terminal part of Mce1A, reacted strongly with GST-Mce1A. The antibody cross-reacted with GST-Mce1F, but not with the other recombinant GST-Mce1 fusion proteins or free GST. Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains. Higher homology was found between the residues 106-165 of Mce1A and the residues 347-406, further into the mature Mce1F polypeptide chain. There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1-5 76C may be narrowed to the KRRITPKD region, the residues 131-138 in Mce1A corresponding to the residues 372-379 in Mce1F. This was confirmed in enzyme-linked immunosorbent assay, showing binding of TB1-5 76C to a 17-mer synthetic peptide containing the KRRITPKD sequence.

Macrophages are a significant source of type 1 cytokines during mycobacterial infection.

T-helper 1 (Th1) cells are believed to be the major producer of the type 1 cytokine interferon-gamma (IFN-gamma) in cell-mediated immunity against intracellular infection. We have investigated the ability of macrophages to release type 1 cytokines and their regulatory mechanisms using both in vivo and in vitro models of pulmonary mycobacterial infection. During pulmonary infection by live Mycobacterium bovis bacilli Calmette-Guérin (BCG) in wild-type mice, lung macrophages released interleukin-12 (IL-12), IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), and expressed surface activation markers. However, macrophages in infected IL-12(-/-) mice released TNF-alpha but not IFN-gamma and lacked surface activation makers. In freshly isolated lung macrophages from naive IL-2(-/-) mice, mycobacteria alone released TNF-alpha but not IFN-gamma, whereas exogenously added IL-12 alone released a minimum of IFN-gamma. However, these macrophages released large quantities of IFN-gamma upon stimulation with both mycobacteria and IL-12. In contrast, mycobacteria and exogenous IFN-gamma released only a minimum of endogenous IFN-gamma. Endogenous IL-18 (IFN-gamma-inducing factor) played little role in IFN-gamma responses by macrophages stimulated by mycobacteria and IL-12. Our data reveal that macrophages are a significant source of type 1 cytokines during mycobacterial infection and that both IL-12 and intracellular pathogens are required for the release of IFN-gamma but not TNF-alpha. These findings suggest that macrophages regulate cell-mediated immunity by releasing not only IL-12 and TNF-alpha but also IFN-gamma and that full activation of IFN-gamma response in macrophages is tightly regulated.

The transferrin binding protein B of Moraxella catarrhalis elicits bactericidal antibodies and is a potential vaccine antigen.

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.

Lack of both types 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin in IL-12-deficient mice.

Understanding of key cytokines and the nature of protective immune responses in pulmonary mycobacterial diseases remains a task of paramount importance. In this study, both wild-type (wt) and IL-12-deficient (IL-12(-/-)) mice were infected by airways inoculation of live Mycobacterium bovis bacille Calmette-Guérin (BCG). The type 1 cytokines IL-12, IFN-gamma, and TNF-alpha, but not the type 2 cytokines IL-4 and granulocyte macrophage (GM)-CSF, markedly increased in the lung and peripheral blood of wt mice postinfection, which resulted in the development of intense granulomatous responses and the effective control of mycobacterial infection in the lung. In contrast, IL-12(-/-) mice demonstrated a lack of both types 1 and 2 cytokines in the lung and blood and a severely impaired tissue immune-inflammatory response lacking not only macrophages and neutrophils but CD4 and CD8 T cells and NK cells in the lung throughout the entire course of study. Total lung mononuclear cells isolated from these mice, in contrast to wt mice, had an impaired recall immune response to Ag challenge in vitro. These impaired responses resulted in an uncontrolled local growth and systemic spread of bacilli. Our findings reveal that IL-12 plays an irreplaceable role in the initiation of Th1 responses, and the loss of its function cannot be compensated for by alternative mechanisms in the lung. This cytokine, together with IFN-gamma and TNF-alpha, and granulomatous inflammation are critically required for the effective control of pulmonary mycobacterial infection. Our results also indicate that the absence of type 1 cytokines does not necessarily favor a Th2 response.

Effect of lipid modification on the physicochemical, structural, antigenic and immunoprotective properties of Haemophilus influenzae outer membrane protein P6.

The outer membrane lipoprotein, P6 of Haemophilus influenzae was studied to determine the importance of the native palmitoyl moiety on its physicochemical and immunological properties. A recombinant P6 (rP6) molecule devoid of lipidation signal sequence was expressed in Escherichia coli and its properties were compared to those of the palmitylated protein purified from H. influenzae. The isoelectric point of rP6 was more acidic than that of the native protein and also exhibited less secondary structure than P6 as judged by circular dichroism. However, both forms of P6 induced identical P6-specific antibody titers in guinea pigs when Freund's adjuvant was used. These antisera reacted with a panel of overlapping P6 peptides in a comparable manner and in addition, rabbit antisera raised against the P6 peptides reacted equally well with P6 and rP6. Furthermore, all human convalescent sera tested exhibited similar anti-P6 and anti-rP6 antibody titers. However, rP6 was less immunogenic than P6 when administered either without adjuvant or in alum and when tested in competitive inhibition studies with anti-P6 antibodies, was a less effective inhibitor than native P6, suggesting a diminution in some of the antigenic activity of rP6. In spite of these differences, rP6 was capable of eliciting a protective antibody response against live H. influenzae type b challenge in a modified infant rat model of bacteremia. These findings demonstrate that the non-fatty acylated rP6 could possibily be substituted for native P6 in a vaccine against H. influenzae.

The major outer membrane protein, CD, extracted from Moraxella (Branhamella) catarrhalis is a potential vaccine antigen that induces bactericidal antibodies.

The major outer membrane protein of Moraxella (Branhamella) catarrhalis, CD, was detergent-extracted from the bacterial cell wall and purified to homogeneity in high yields by a simple process. The purified protein appeared to exhibit immunogenic properties similar to those of native CD exposed on the surface of the bacterium. Antibodies to CD raised in mice specifically bound to intact B. catarrhalis, as determined by flow cytometry analysis. The IgG subclass distributions of anti-CD antibodies in sera from mice immunized with purified CD or with B. catarrhalis were also similar. CD was found to be antigenically conserved among a panel of B. catarrhalis isolates, as demonstrated by the consistent reactivities of mouse anti-CD antisera with a common 60 kDa protein on immunoblots. Furthermore, convalescent sera collected from patients with otitis media due to B. catarrhalis infection were found to be reactive with the CD protein by immunoblotting. Finally, the purified protein induced antibodies in guinea pigs and mice that exhibited in vitro bactericidal activity against the pathogen. Therefore, the native CD outer membrane protein represents a potentially useful antigen for inclusion in a vaccine against B. catarrhalis.

Is post-hypoxic-ischemic cell damage associated with excessive ATP consumption rather than a failure of ATP production?

Secondary cell damage after ATP depletion due to hypoxia or ischemia is clinically important because it correlates with residual effects; post-hypoxic-ischemic fits can be associated with later cerebral palsy. The mechanisms involved in delayed secondary cell damage are not clear, possibly because extensive relevant evidence is often fragmented. However, a sequence of changes can be suggested; this cross-linked sequence is tentatively outlined in this review. The outline suggests explanations for otherwise ill-understood clinical disturbances such as the loss of inhibitory control in damaged cells and the well documented reduction of cellular ATP. Loss of control may be due to reduced synthesis of control proteins and the reduced ATP concentration may be due to increased energy consumption.

The importance of the measurement of ATP depletion and subsequent cell damage with an estimate of size and nature of the market for a practicable method: a review designed for technology transfer.

ATP is the energy currency of cells. ATP depletion is a central process in pathogenesis, in particular ischaemia, hypoxia and hypoglycaemia. ATP depletion in cells can be indirectly measured from the increased concentrations of extracellular hypoxanthine, a central intermediate in the metabolism of ATP. Cell damage secondary to ATP depletion can also be measured from extracellular hypoxanthine. The relevant biochemistry and physiology is briefly reviewed. Since market size is needed for investment decisions that would allow technology transfer, the numbers of hypoxanthine analyses that are clinically justified from the extensive published evidence are calculated per million population from UK, Norwegian and other evidence. The concentration of oxygen in blood is measured to estimate whether mitochondrial oxidative phosphorylation is adequate. Measurements of bicarbonate are used to estimate anaerobic glycolysis. Since the indirect estimation of ATP depletion is a major objective of blood gas and acid-base analyses, the number of such analyses per million population provides a good estimate of potential market size for a more direct method of estimating ATP depletion. A method is required for the rapid, dispersed emergency analyses needed clinically. Routes for method development are indicated. Competition, risks, acceptability, consumer motivation and timetables are indicated for the development phase. There are medicolegal pressures, especially in the USA, for the proposed advances to be widely used.

Cloning and expression of the Haemophilus influenzae transferrin receptor genes.

The genomic transferrin receptor genes (tbpA and tbpB) from two strains of Haemophilus influenzae type b (Hib) and two strains of non-typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95-100% conserved and those of the tbpB genes were 66-100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N-terminal sequences of Tbp1 and Tbp2 and were found to encode full-length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti-rTbp2 antiserum, but not after anti-rTbp1 antiserum.

Lesch-Nyhan syndrome and its pathogenesis: normal nicotinamide-adenine dinucleotide but reduced ATP concentrations that correlate with reduced poly(ADP-ribose) synthetase activity in HPRT-deficient lymphoblasts.

In hypoxanthine (guanine) phosphoribosyltransferase- (HPRT; EC 2.4.2.8) deficient lymphoblasts, ATP but not nicotinamide-adenine dinucleotide coenzyme concentrations are reduced by limited nutrition. Such reduced ATP concentrations are correlated with reduced poly(ADP-ribose) synthetase (polyADPRT; EC 2.4.2.30) activity; this reduces the breakdown of nicotinamide-adenine dinucleotide coenzymes and thus explains their normal intracellular concentrations. Since reductions in poly(ADP-ribose) synthetase activity reduce DNA repair, alterations in DNA could accumulate even in non-multiplying cells such as neurons, especially in the continuously active 'respiratory centre'. Our Lesch-Nyhan patients suffered respiratory deaths between 15 and 20 years of age.

Branhamella catarrhalis pathogenesis in SCID and SCID/beige mice. Brief report.

SCID and SCID/beige mice were used to study the pathogenesis of B. catarrhalis administered by intranasal, intraperitoneal or intravenous routes. Challenged adult animals did not appear overtly clinically ill. Similar symptoms were observed regardless of the challenge route, and pretreatment of mice with human transferrin did not enhance clinical virulence. Susceptibility to B. catarrhalis appeared to be age-dependent as some mice under one week of age died following challenge. Postmortem findings included circumscribed pale foci on the liver, splenomegaly and mineralization of the myocardium. Presence of lesions did not correlate with the assessment of clinical well being, and severity of the lesions was found to be challenge strain-dependent. Liver lesions and splenomegaly were not observed in animals challenged with heat-killed bacteria or placebo. SCID/beige mice were more affected than SCID mice both clinically and pathologically, suggesting that natural killer cell and polymorphonuclear cell functions may be important in resolving B. catarrhalis challenge.

Identification of surface-exposed B-cell epitopes recognized by Haemophilus influenzae type b P1-specific monoclonal antibodies.

A panel of P1 synthetic peptides was synthesized to map the surface-exposed epitopes of Haemophilus influenzae type b outer membrane protein P1 recognized by three murine monoclonal antibodies (MAbs 7C8, 3E12, and 6B1). By using peptide-specific enzyme-linked immunosorbent assays, MAbs 6B1, 7C8, and 3E12 were shown to recognize distinct epitopes localized within residues 60 to 88, 165 to 193, and 400 to 437 of mature P1, respectively. Since MAb 7C8 was shown previously to be protective against certain H. influenzae type b subtypes in the infant rat model of bacteremia, its cognate epitope was further characterized by using truncated peptide analogs. Fine mapping of the 7C8 epitope by competitive inhibition studies revealed that it was localized within residues 184 and 193.

Introduction to the age-related diagnosis (ARD) index: an age at presentation related index for diagnostic use.

Development and, therefore, age is important in paediatrics. Diagnostically useful data from this journal has been related to age and organized to form an age-related diagnostic (ARD) index. The ARD index is designed for non-expert clinical and laboratory workers to use in the early phases of diagnosis and as an addition to existing diagnostic schemes. Entry to the index is from the age at clinical presentation. Each entry is a sequence starting with clinical and laboratory presentations, clinical course, laboratory key investigations and finally diagnosis with volume and page numbers of the original article, the primary source. Within age groups, entries are grouped by diagnoses with the commonest diagnosis first; this has the effect of roughly but not precisely grouping similar clinical and laboratory findings.