Development of intra-natural killer complex (NKC) recombinant and congenic mouse strains for mapping and functional analysis of NK cell regulatory loci.
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Animals↗
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Design of cassette baculovirus vectors for the production of therapeutic antibodies in insect cells.
BACKGROUND: Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. OBJECTIVES: To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. STUDY DESIGN: Two transfer vectors, pBHuC kappa and pBHuC gamma 1, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain C kappa gene or heavy chain C gamma 1 sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. RESULTS: Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. CONCLUSION: Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.
Animals↗
Biological evaluation and drug delivery application of cationically modified phospholipid polymers.
Phospholipid-like polymers based on 2-methacryloyloxyethyl phosphorylcholine containing varying amounts of the cationically charged monomer choline methacrylate (CMA) from 0 to 30 wt% have been prepared. Substrates coated with these materials were shown to bind significantly lower amounts of specific proteins compared to the uncoated control. ELISA assays demonstrated that fibrinogen did not bind appreciably to coatings containing 0-30% CMA, whereas albumin binding was seen to increase significantly as the CMA content of the coating increased. Platelet activation assays, measurement of plasma coagulation time and whole blood contact scanning electron micrography demonstrated that the haemocompatibility of the coatings was shown to be unaffected by the CMA component. The CMA polymer coatings have been shown to absorb/adsorb many different drug compounds covering a wide range of molecular weights and release these in a controlled fashion. The range of cationic polymers assessed can interact with the net negative charge found in many large therapeutic biomolecules, such as DNA fragments used in gene therapy, that may be of interest in the preventative treatment of conditions such as restenosis. Coronary stents coated with 6% or 20% CMA-containing polymers have been shown to load and release this type of genetic material irrespective of molecular weight of the biomolecule. Ex vivo and in vivo studies have shown that these compounds can be delivered to the stented section of the vessel with very low quantities delivered outside the vessel target area.
Absorption↗
SARS-CoV-2 genomic diversity and within-host evolution in individuals with persistent infection in the UK: an observational, longitudinal, population-based surveillance study.
BACKGROUND: Persistent SARS-CoV-2 infections in hospitalised immunocompromised individuals are known to facilitate accelerated within-host viral evolution, potentially contributing to the emergence of highly divergent variants. However, little is known about the evolutionary dynamics and transmission risks of persistent infections in the general population. We aimed to characterise the within-host evolution of SARS-CoV-2 during persistent infections identified through a large community surveillance study. METHODS: We used data from the Office for National Statistics COVID-19 Infection Survey (ONS-CIS), a large-scale, longitudinal, population-based surveillance study conducted in the UK from April, 2020, to March, 2023. For this analysis, we focused on infections with high viral load (cycle threshold ≤30) and available genome sequences, from seven major SARS-CoV-2 lineages (alpha, delta, BA.1, BA.2, BA.4, BA.5, and XBB). ONS-CIS participants were randomly selected from the general population and tested regularly by RT-PCR, regardless of symptoms. We defined persistent infections as those with sustained or rebounding high viral RNA titres for 26 days or longer. We examined associated host characteristics and used raw sequence data to identify de novo mutations and estimate within-host synonymous and non-synonymous evolutionary rates across the SARS-CoV-2 genome. FINDINGS: Between Nov 2, 2020, and March 21, 2023, we identified 576 persistent infections with at least two sequences, including 11 alpha, 106 delta, 102 BA.1, 204 BA.2, 16 BA.4, 133 BA.5, and 4 XBB. Persistent infections were more common in males than females (p<0·0001) and individuals older than 60 years (p=0·0027). The median within-host genome-wide evolutionary rate was 7·9 × 10-4 substitutions per site per year (IQR 7·0-9·0 × 10-4), with high inter-individual variability driven largely by non-synonymous mutations, particularly in the N-terminal and receptor-binding domains of the spike protein. Longer infection duration was associated with higher evolutionary rates, while no associations were found with age, sex, vaccination status, previous infection, or virus lineage. We found no clear evidence of transmission beyond the first month of infection in any of the 84 persistent infections lasting 56 days or longer. In total, we identified 379 recurrent mutations, including many with known or predicted negative fitness effects and low prevalence at the population level, as well as de novo reversions to the Wuhan-Hu-1 reference sequence, which were likely under positive selection within those individuals. INTERPRETATION: This study highlights the heterogeneous nature of within-host SARS-CoV-2 evolution in individuals with persistent infection in the community. Notably, a small subset of persistent infections with high viral loads underwent accelerated viral evolution or recurrently acquired hallmark mutations found in novel variants. In addition, onward transmission from a persistent infection during the later stages of infection is likely to be rare. These insights have important implications for prioritising genomic surveillance and managing patients with persistent infections. FUNDING: Department of Health and Social Care.
Humans↗
Evidence for negative selective pressure in HIV-2 evolution in vivo.
HIV-2 sequence divergence and evolution in vivo has not been well characterized so far. To investigate the extent of HIV-2 genetic diversity and better understand how HIV-2 evolves in vivo, env C2-C3 nucleotide sequences were obtained from the plasma and PBMCs virus populations of four HIV-2 patients with different infection periods. Phylogenetic analysis showed that three patients were infected with subtype A HIV-2 and the remaining patient was infected with a divergent HIV-2 that could not be genotyped. Virus populations from the plasma and PBMCs clustered together in all patients suggesting that there is continuous and unrestricted virus flow between plasma and PBMCs. HIV-2 genetic diversity was not correlated with CD4+ cell counts and plasma viral load. There was a direct association between the period of infection and genetic divergence of virus populations both in the env C2-C3 and V3 regions such that higher genetic diversity was observed in long-term infected patients. In three patients, the average frequency of synonymous substitutions (dS) was significantly higher than the nonsynonymous substitutions (dN) whereas in the fourth patient the dN/dS ratio approached the unity. These data demonstrate that negative selective pressure determines the evolution of the HIV-2 env C2-C3 region in vivo. Our results suggest that throughout HIV-2 infection low virus adaptation to strong selective pressures (e.g. immune pressure) promotes the predominance of a few optimally adapted forms.
Amino Acid Sequence↗
A mathematical model of the dynamics of scrapie in a sheep flock.
A mathematical model is developed for the dynamics of an outbreak of scrapie in a single sheep flock with the aim of assisting the interpretation of field data. The model incorporates age structure of the sheep population, both horizontal and vertical transmission, genetic predisposition to infection, variable initial load of the infectious agent, and increasing infection load during an incubation period of the same order as sheep life expectancy. This leads to system of partial differential equations with respect to time, age and infection load. Numerical analyses using this model demonstrate that a scrapie outbreak is likely to be of long duration (several decades), will lead to reduction of scrapie susceptible allele frequency (but not to zero), and has different dynamics in homozygous and heterozygous susceptible sheep, even if these genotypes are equally susceptible, due to the different contributions of vertical infection to transmission to genotypes.
Age Factors↗
Sustained phenotypic correction in a mouse model of hypoalphalipoproteinemia with a helper-dependent adenovirus vector.
We examined the efficacy and host response to the adenovirus (Ad)-mediated delivery of human apolipoprotein A-I (APOA1) gene to the liver of APOA1(-/-) mice. Administration of a first-generation vector (FGAd-AI) resulted in a transient appearance of APOA1 in plasma and induced an anti-APOA1 antibody titer, whereas treatment with a helper-dependent vector (HDAd-AI) resulted in sustained APOA1 expression without inducing an antibody titer. With these results, we studied the effects of FGAd vectors on APOAI expression by HDAd-AI vector. Co-treatment with an FGAd vector inhibited HDAd-AI- mediated APOA1 expression independent of transgene cassettes, but only FGAd-AI induced a humoral response. Furthermore, APOA1 mRNA levels in mice co-treated with FGAd vectors were much lower than those expected from the vector copy number, suggesting that DNA of FGAd vectors interferes with the HDAd-AI vector's APOA1 promoter. A single treatment with an HDAd-AI vector produced a supraphysiological plasma APOA1 level that gradually declined to about half the normal human level over the course of 2 years, associated with a plasma cholesterol level that is persistently higher than that in controls. This investigation provides the proof of principle that liver-directed HDAd gene delivery is effective for the long-term phenotypic correction of monogenic hypoalphalipoproteinemia.
Adenoviridae↗
Polyriboinosinic polyribocytidylic acid [poly(I:C)]/TLR3 signaling allows class I processing of exogenous protein and induction of HIV-specific CD8+ cytotoxic T lymphocytes.
In the case of viral infection, various viral proteins and genetic components are disseminated in the body. The former viral proteins may be captured by immature dendritic cells (DC) and the latter genetic components may stimulate the antigen-loading DC to maturate via specific Toll-like receptors (TLR), leading to the establishment of virus-specific cellular immunity; in particular, cytotoxic T lymphocytes (CTL) that control intracellular virions. Polyriboinosinic polyribocytidylic acid [poly(I:C)], which might reflect a natural genetic product from a variety of viruses during replication, has recently been identified as one of the critical stimuli for TLR3. Based on these observations, we speculated that stimulation of TLR3 with poly(I:C) might drive the direction of acquired/adaptive immunity to the cellular arm. Indeed, when BALB/c mice were immunized with purified recombinant HIV-1 envelope gp120 or influenza hemagglutinin (HA) protein together with poly(I:C), epitope-specific CD8(+) class I MHC molecule-restricted CTL were primed from naive CD8(+) T cells in vivo. In contrast, when the same proteins were immunized with lipopolysaccharide, a stimulant of TLR4, specific CTL were not primed at all. Moreover, we show here that immature DC could present processed antigen from captured purified protein in association with class I MHC molecules in the presence of poly(I:C), but not of LPS. These results indicate that we are able to manipulate the direction of acquired/adaptive effector immune responses using an appropriate stimuli and the findings presented in this paper will offer a new therapeutic strategy using poly(I:C) administration for priming antigen-specific CD8(+) CTL with purified viral protein in vivo.
Animals↗
Sodium balance and peripheral resistance in arterial hypertension.
In this brief review, some aspects of the relation between sodium balance and peripheral resistance in four types of arterial hypertension are discussed. Renal hypertension, hypertension caused by reduced kidney mass and sodium load, mineralocorticoid hypertension, and spontaneous or "genetic" hypertension in rats are considered. In all these forms of hypertension, the increase in peripheral resistance is preceded by changes in body sodium and fluids. However, the precise role of these changes in the development of the subsequent rise of peripheral resistance is not yet clear. Changes in ion transport across the cell membranes have been demonstrated even before the development of hypertension, especially the forms caused by mineralocorticoids or genetic factors. Even though we do not know the underlying mechanisms, it is very likely that these cell-membrane changes are involved in the rise of peripheral resistance and blood pressure.
Animals↗
Hierarchies of life histories and associated health risks.
Widely documented inverse associations between socioeconomic standing and incident chronic disease and mortality invite explanation in terms of pathways to these outcomes. Empirical identification of pathways, or histories, requires measures that assess cumulative wear and tear on physiological systems following from psychosocial adversity and genetic predispositions. Such an assessment, allostatic load, has been shown to predict later life mortality, incident cardiovascular disease, and decline in physical and cognitive functioning. Using data from the Wisconsin Longitudinal Study (WLS), we seek precursors to allostatic load via ordered categories of cumulative adversity relative to advantage over the life course. We operationalize these histories via unfolding economic circumstances and social relationship experiences (e.g., parent-child interactions, quality of spousal ties). Findings reveal a strong direct association between the extent of adversity relative to advantage in an ordering of these histories and likelihood of high allostatic load. Importantly, resilient individuals with economic disadvantage, but compensating positive social relationship histories also show low prevalence of high allostatic load.
Cardiovascular Diseases↗
The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase.
Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.
Amino Acid Sequence↗
Hepatitis B virus genome mutations in precore and basal core promoter regions among HBeAg-negative chronic hepatitis B patients with high viral load in Indonesia.
Hepatitis B e antigen (HBeAg) is widely used as a marker for active HBV replication and serves as a surrogate for HBV DNA > 200,000 IU/mL to determine eligibility for tenofovir disoproxil fumarate (TDF) prophylaxis to prevent vertical transmission, according to WHO guidelines. However, some HBeAg-negative patients still harbor high viral loads. Mutations in the precore (PC) and basal core promoter (BCP) regions may reduce or abolish HBeAg expression without necessarily suppressing viral replication. Next-generation sequencing (NGS)-based characterization of these mutations remains limited in Indonesia. This study aimed to analyze the mutation prevalence in the BCP and PC regions associated with HBeAg negativity in Indonesian patients. We conducted a cross-sectional study of 32 chronic HBV treatment-naïve, unvaccinated patients with HBV DNA > 200,000 IU/mL (16 HBeAg-negative, 16 HBeAg-positive) at Cipto Mangunkusumo General Hospital. BCP and PC mutations were analyzed using NGS, classifying mutations as major (mutation frequency index [MFI] ≥20%) or minor (MFI 1- < 20%). Associations were analyzed using the Chi-square or Fisher's exact test and p-values were adjusted using the Benjamini-Hochberg procedure. Among 29 major mutation sites, PC mutations A1846T/C and G1896A were more frequent in HBeAg-negative than HBeAg-positive patients (81.3% vs 6.3% and 75.0% vs 12.5%, respectively; all adjusted p = 0.019). Combined analysis showed higher mutation frequencies in HBeAg-negative patients (93.8%, 81.3%, and 62.5% for A1846T/C, G1896A, and G1899A, respectively; all adjusted p = 0.015). In conclusion, HBeAg-negative patients with high viral loads are strongly associated with PC mutations, particularly G1896A, A1846T/C, and G1899A. These exploratory findings provide regional NGS-based molecular evidence that established PC mutations may contribute to the coexistence of HBeAg negativity and continued high-level HBV replication in Indonesian patients. Larger studies incorporating broader virological and clinical comparison groups are required to determine the clinical significance of these findings.
Humans↗
Idiopathic slipped capital femoral epiphysis in Amish children.
BACKGROUND: Of the many studies of slipped capital femoral epiphysis, none have specifically addressed Amish children. The Amish reflect a small gene pool relative to the general white North American population. Additional knowledge of the demographics of this disorder in Amish children may provide genetic insights. The purpose of this study was to review the demographics of slipped capital femoral epiphysis in the Amish population. METHODS: A retrospective review of the cases of twenty-five Amish children with slipped capital femoral epiphysis treated at two institutions was performed. The child's gender, age, weight, height, and body mass index at the time of the diagnosis; duration of symptoms; laterality of the slip; birth weight; family history; and slip severity were recorded. The slip was classified as stable or unstable. Patients who had been included in a previously published multicenter study served as a control group. RESULTS: There were seventeen boys and eight girls with a total of thirty-three slipped capital femoral epiphyses; eight of the slips were bilateral. At the time of the diagnosis, the mean age (and standard deviation) was 13.4 +/- 1.6 years, the mean weight and height were 55.6 +/-12.4 kg and 155.5 +/- 10.2 cm, and the mean body mass index was 23.4 +/- 5.4 kg/m(2). The mean duration of symptoms was 6.6 +/-9.0 months. There were thirty-one stable and two unstable slips with a mean slip angle of 38 degrees +/- 20 degrees . Nine (39%) of twenty-three children for whom the information had been recorded had a positive family history of slipped capital femoral epiphysis, a rate that is higher than the 9% and 14.5% rates reported in two other series (p = 0.002). The Amish children were not as heavy as their non-Amish counterparts (55.6 +/- 12.4 kg compared with 66.4 +/- 17.7 kg, p = 0.0036). CONCLUSIONS: Although the children in this study were moderately heavy, they could not be classified as obese on the basis of weight-for-age or body-mass-index percentiles. The high prevalence of family members with slipped capital femoral epiphysis may reflect either a genetic or environmental component, or an interaction between genetics and environment (for example, work load or common chores requiring particular physical positions) in the Amish population.
Body Height↗
Understanding HIV-1 viral load.
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Biomarkers↗
Heterozygosity for the H63D mutation in the hereditary hemochromatosis (HFE) gene may lead into severe iron overload in beta-thalassemia minor: observations in a thalassemic kindred.
Heterozygosity for beta-thalassemia (minor) by itself does not lead into iron overload; however, when it is inherited together with a homozygous state for either the H63D or the C282Y mutations of the hereditary hemochromatosis gene (HFE gene), iron overload may ensue. We describe here a kindred in which the propositus, being heterozygote for beta-thalassemia and the H63D mutation of the HFE gene, developed severe iron overload and in turn, chronic liver failure with portal hypertension. Other members of the family with either beta-thalassemia or heterozygous for the H63D gene mutation did not develop iron overload. The interaction between beta-thalassemia and hereditary hemochromatosis is briefly discussed and speculations about other possible genetic mutations leading into familial iron loading are done.
Adult↗
Encapsulated cell-based delivery of CNTF reduces photoreceptor degeneration in animal models of retinitis pigmentosa.
PURPOSE: The objective of the present study was to evaluate the therapeutic efficacy of ciliary neurotrophic factor (CNTF) delivered through encapsulated cells directly into the vitreous of the eye in an rcd1 canine model of retinitis pigmentosa. The dose-range effect of the treatment was also investigated. METHODS: Polymer membrane capsules (1.0 cm in length and 1.0 mm in diameter) were loaded with mammalian cells that were genetically engineered to secrete CNTF. The cell-containing capsules were then surgically implanted into the vitreous of one eye of rcd1 dogs at 7 weeks of age, when retinal degeneration is in progress but not complete. The contralateral eyes were not treated. The capsules remained in the eyes for 7 weeks. At the end of the studies, the capsules were explanted, and CNTF output and cell viability were evaluated. The eyes were processed for histologic evaluation. RESULTS: In each animal, the number of rows of photoreceptor nuclei in the outer nuclear layer (ONL) was significantly higher in the eye that received a CNTF-secreting implant than in the untreated contralateral eye. No adverse effects were observed on the retina in the treated eyes. The explanted capsules produced a low level of CNTF. The cells in the capsules remained viable and densely distributed throughout. CONCLUSIONS: CNTF delivered through encapsulated cells directly into the vitreous of the eye protects photoreceptors in the PDE6B-deficient rcd1 canine model. Furthermore, sparing of photoreceptors appeared dose-dependent with minimum protection observed at CNTF doses of 0.2 to 1.0 ng/d. Incrementally greater protection was achieved at higher doses. The surgically implanted, cell-containing capsules were well tolerated, and the cells within the capsule remained viable for the 7-week implantation interval. These results suggest that encapsulated cell therapy may provide a safe and effective strategy for treating retinal disorders in humans.
Animals↗
Neuromuscular aspects of osteoarthritis: a perspective.
Osteoarthritis (OA) represents failure of the diarthrodial joint and may be due to a primary abnormality in any of the tissues of the joint, e.g. articular cartilage, subchondral bone, synovium, periarticular muscle, or sensory nerves whose termini lie within the joint. Neuropathic arthropathy, due to severe sensory neuropathy, causes severe and rapid breakdown of joints. We have shown that interruption of sensory input from the ipsilateral hind limb strikingly accelerates progression of OA after anterior cruciate ligament transection in the dog; a clinical correlate exists in humans with diabetic neuropathy who sustain even minor joint trauma. Knee OA in humans is accompanied by defects in proprioception, although it is not clear whether the neurological abnormality is primary or a consequence of intra-articular pathology. The magnitude of the load on a joint and, especially, the rate of impulsive loading, influence development of OA. It is relevant, therefore, that quadriceps weakness may precede development of knee OA in some people, insofar as this may diminish the effectiveness of protective muscular reflexes and thereby increase deleterious joint loading. Individuals vary with respect to how they load their joints, perhaps because of genetic differences in central program generators.
Animals↗
Haptoglobin polymorphism, iron metabolism and mortality in HIV infection.
BACKGROUND: Three phenotypes of the antioxidant protein haptoglobin are known: Hp 1-1, Hp 2-1 and Hp 2-2. OBJECTIVES: To investigate the outcome of HIV infection according to haptoglobin type. DESIGN AND METHODS: Haptoglobin phenotypes were determined using starch gel electrophoresis in serum obtained from 653 HIV-infected Caucasians in the AIDS reference centers of Gent (n = 184), Antwerp (n = 309), and Luxembourg (n = 160). Survival was compared between haptoglobin types using Kaplan-Meier curves. Plasma HIV-1 RNA was quantified by reverse transcriptase PCR. Serum iron, transferrin saturation, ferritin, and vitamin C were assayed to evaluate iron-driven oxidative stress in 184 HIV-infected patients and 204 controls. RESULTS: The haptoglobin type distribution amongst the patients (17.6% Hp 1-1, 49.9% Hp 2-1, 32.5% Hp 2-2) corresponded to that of the controls. Kaplan-Meier curves showed a higher mortality for the Hp 2-2 group (P = 0.0001; adjusted mortality risk ratio, 1.78; 95% confidence interval, 1.25-2.54). Median survival time was 11.0 years (Hp 1-1 and Hp 2-1) versus 7.33 years (Hp 2-2). Plasma HIV-1 RNA levels prior to antiviral therapy and their increase over 1 year were highest in Hp 2-2 patients (P = 0.03 and 0.003, respectively). The Hp 2-2 type was associated with higher serum iron, transferrin saturation, and ferritin levels and with low vitamin C concentrations. Furthermore, ferritin concentrations were higher in HIV-infected patients than in controls (P < 0.0001). CONCLUSION: HIV-infected patients carrying the Hp 2-2 phenotype show a worse prognosis, which is reflected by a more rapid rate of viral replication (in the absence of antiviral treatment). They also accumulate more iron and oxidize more vitamin C, suggesting that less efficient protection against haemoglobin/iron-driven oxidative stress may be a direct mechanism for stimulating viral replication.
Adult↗