An effective CTL peptide vaccine for Ebola Zaire Based on Survivors' CD8+ targeting of a particular nucleocapsid protein epitope with potential implications for COVID-19 vaccine design.

The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple Class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus.

Prevalence of complementary and alternative medicine (CAM) use by the general population: a systematic review and update.

OBJECTIVES: To update previous systematic reviews of 12-month prevalence of complementary and alternative medicine (CAM) use by general populations; to explore trends in CAM use by national populations; to develop and apply a brief tool for assessing methodological quality of published CAM-use prevalence surveys. DESIGN: Nine databases were searched for published studies from 1998 onwards. Studies prior to 1998 were identified from two previous systematic reviews. A six-item literature-based tool was devised to assess robustness and interpretability of CAM-use estimates. RESULTS: Fifty-one reports from 49 surveys conducted in 15 countries met the inclusion criteria. We extracted 32 estimates of 12-month prevalence of use of any CAM (range 9.8-76%) and 33 estimates of 12-month prevalence of visits to CAM practitioners (range 1.8-48.7%). Quality of methodological reporting was variable; 30/51 survey reports (59%) met four or more of six quality criteria. Estimates of 12-month prevalence of any CAM use (excluding prayer) from surveys using consistent measurement methods showed remarkable stability in Australia (49%, 52%, 52%; 1993, 2000, 2004) and USA (36%, 38%; 2002, 2007). CONCLUSIONS: There was evidence of substantial CAM use in the 15 countries surveyed. Where national trends were discernable because of consistent measurement, there was no evidence to suggest a change in 12-month prevalence of CAM use since the previous systematic reviews were published in 2000. Periodic surveys are important to monitor population-level CAM use. Use of government-sponsored health surveys may enhance robustness of population-based prevalence estimates. Comparisons across countries could be improved by standardising approaches to data collection.

Neurofunctional imaging of ß-cell dynamics.

Here, we outline how islet cells use autocrine and paracrine 'circuits' of classical neurotransmitters and their corresponding receptors and transporters to communicate with vicinal ß-cells to regulate glucose-stimulated insulin secretion. Many of these same circuits operate in the central nervous system and can be visualized by molecular imaging. We discuss how these techniques might be applied to measuring the dynamics of ß-cell function in real time.

The GnRH test in the assessment of patients with pituitary and parapituitary lesions: results of a 5-year retrospective study.

OBJECTIVE: To examine the utility of the GnRH (gonadotrophin-releasing hormone) test in the management of patients with pituitary and parapituitary lesions. PATIENTS AND METHODS: A 5-year retrospective study of LH (luteinizing hormone) and FSH (follicle stimulating hormone) responses to GnRH test in patients with HP (hypothalamic-pituitary) disease in a regional endocrine centre. Serum LH and FSH concentrations were measured at baseline and at 20 and 60 min after an intravenous bolus of 100 mcg (micrograms) of GnRH. The GnRH responses were categorised by tumour size, tumour type, and gonadal status. RESULTS: Of the 104 patients studied, 46 were male and 58 were female. There were 50 normal, 38 subnormal and 16 exaggerated LH responses compared with 34 normal 67 subnormal and three exaggerated responses for FSH. Seventy-four patients (71.2%) were hypogonadal. Normal LH responses were achieved in half of the hypogonadal subjects and normal FSH responses in more than a third. Furthermore, the LH responses were exaggerated in nine hypogonadal patients compared with three for FSH. The GnRH test could not differentiate between pituitary or parapituitary lesions either by size or type of lesion. An exception was the male non-functioning adenoma (NFA) sub-group (10 patients, all were hypopituitary, seven were hypogonadal), which demonstrated significant subnormal LH and FSH responses compared with other male and female tumour type sub-groups. CONCLUSIONS: The data from this study indicate that the GnRH test is unhelpful in the clinical assessment of the HP axis in patients with HP disease.

The treatment of de novo acromegalic patients with octreotide-LAR: efficacy, tolerability and cardiovascular effects.

AIM: Somatostatin analogues are normally used as adjunctive therapy to surgery and radiotherapy in management of acromegaly. We studied the effects of de novo OCT-LAR treatment on growth hormone (GH) suppression, tumour size, cardiovascular function, clinical symptoms, signs and quality of life in 9 newly diagnosed acromegalic patients. METHODS: Patients commenced OCT-LAR 20 mg IM monthly for 2 months. Dose increased to 30 mg monthly if mean serum GH (MGH) > 5 mU/l (2 microg/litre) (7 patients). Treatment continued for 6 months. Cardiac function assessed by echocardiography at baseline and day 169. Left ventricular (LV) mass and ejection fraction (EF) calculated from 2D M-mode studies. RESULTS: Serum GH demonstrated suppression in 8/9 patients (mean suppression 64.9% +/- 29.7%, range; 4-95.2%). MGH suppressed < 5 mU/ (2 microg/litre) in 3 (33%) patients. IGF-I and IGFBP3 normalised in 1 (12.5%) and 3 (38%) patients respectively. Tumour shrinkage seen in 30% patients. Eight patients were assessed by echocardiography. At baseline, 7 patients demonstrated abnormalities in LV mass and EF. At day 169, 6 patients demonstrated a fall and 1 an increase in LV mass. Overall there was no significant change in LV mass. A significant increase in EF was observed (p = 0.02). There were significant improvements in health perception (p = 0.01), fatigue (p < 0.05) and perspiration (p = 0.0039). CONCLUSIONS: These data demonstrate OCT-LAR provides adequate control of acromegaly in a proportion of patients treated over 6 months. This is associated with improved LV function, evidenced by increased EF. Improved results are expected with longer-term treatment. OCT-LAR may be considered as primary treatment for acromegaly in selected patients.

Primary medical therapy for acromegaly: an open, prospective, multicenter study of the effects of subcutaneous and intramuscular slow-release octreotide on growth hormone, insulin-like growth factor-I, and tumor size.

Conventional surgery and radiotherapy for acromegaly have limitations. There are few data on the use of the somatostatin analog octreotide (Oct) as primary medical therapy. An open prospective study of 27 patients with newly diagnosed acromegaly was conducted in nine endocrine centers in the United Kingdom. Twenty patients had macroadenomas, and 7 had microadenomas. For the first 24 wk (phase 1), patients received sc Oct in an initial dose of 100 microg, 3 times daily, increased to 200 micro g three times daily after 4 wk in the 13 patients whose mean serum GH remained greater than 5 mU/liter (2 microg/liter). Five-point GH profiles were performed at 0, 4, 12, and 24 wk, and high resolution pituitary imaging using a standard protocol was performed at 0, 12, and 24 wk (magnetic resonance imaging in 25 patients and computed tomography in 2). Tumor dimensions and volumes were calculated by a central, reporting neuroradiologist, and the results were audited by a second, independent neuroradiologist. After 24 wk, 15 patients proceeded to phase 2 of the study with a direct switch to monthly injections of the depot formulation of Oct, Sandostatin long-acting release (Oct-LAR). Further GH profiles were performed at 36 and 48 wk, and pituitary imaging was performed at 48 wk. The median pretreatment serum GH concentration was 30.7 mU/liter (range, 6.7-141.4). During sc Oct, serum GH fell to less than 5 mU/liter in 9 patients (38%), and IGF-I fell to normal in 8 patients (33%). All 27 tumors shrank during sc Oct; for microadenomas the median tumor volume reduction was 49% (range, 12-73), and for macroadenomas it was 43% (range, 6-92). After 24 wk of Oct-LAR (end of phase 2), the GH level was less than 5 mU/liter in 11 of 14 patients (79%), and IGF-I was normal in 8 of 15 patients (53%). In the 15 patients given Oct-LAR (10 macroadenomas), wk 48 scans showed a further overall median tumor volume reduction of 24%. At the end of the study 79% of patients had mean serum GH levels below 5 mU/liter, 53% had normal IGF-I levels, and 73% showed greater than 30% tumor shrinkage. Twenty-nine percent of patients achieved all 3 targets, but no patient with pretreatment GH levels above 50 mU/liter did so at any stage of the study. Primary medical therapy with Oct offers the prospect of normalization of GH/IGF-I levels together with substantial tumor shrinkage in a significant subset of acromegalic patients. This is most likely to occur in patients with pretreatment GH levels less than 50 mU/liter (20 microg/liter).

Hemochromatosis mutations C282Y and H63D in 'cis' phase.

Homozygosity for the C282Y mutation of the HFE gene is a highly significant risk factor for the development of hereditary hemochromatosis (HH) and the majority of patients with HH have this genotype. An Irish/Belgian female with an elevated serum ferritin level and a family history of hemochromatosis was tested for the presence of the C282Y and H63D mutations. Results of digested PCR products have shown the patient to be homozygous for C282Y mutation and heterozygous for H63D mutation. Sequencing confirmed these findings. Genotyping of the patient's offspring and husband has also indicated the inheritance of both C282Y and H63D in 'cis'. Implications of this finding are: 1) the compound heterozygous state is by far the most common, but not the universal, phase for individuals found to be heterozygous for the two mutations, C282Y and H63D; 2) the C282Y and H63D mutations in the 'cis' phase may account for some cases of questionable parentage.

Transcript profiling of human dendritic cells maturation-induced under defined culture conditions: comparison of the effects of tumour necrosis factor alpha, soluble CD40 ligand trimer and interferon gamma.

Using cDNA arrays, we characterized patterns of gene expression in populations of human dendritic cells (DCs) produced for clinical use. Culture and maturation induction of myeloid adherent cells under serum-free conditions yielded DCs with phenotypes similar to those described in serum-based systems. Analysis of gene expression in DCs treated with tumour necrosis factor alpha, soluble CD40L trimer or interferon gamma, however, showed specific patterns for each factor examined. Our studies document the expression of several transcripts that have not hitherto been described in DCs and/or differentially regulated according to the differentiation state of the DCs, and suggest important functional differences among the DC populations examined. In addition, DC maturation directs changes in the levels of mRNA specific for transcriptional regulators that effect the production of cytokines (e.g. BCL-6, c-rel). Other changes observed, including alteration in the gene expression profile of adhesion molecules and chemokine receptors such as CD44H, CD 49B, Rantes R, CXCR5 and CD37, suggest differences in trafficking potential between the populations studied. This broad-based description of DC populations, produced under serum-free conditions, has enabled us to better define intermediate stages of DC maturation as well as the differentiation-inducing effects of cytokines on these cells.

Cushing's syndrome associated with a chemodectoma and a carcinoid tumour.

We present a case of Cushing's syndrome where 111In-octreotide scanning provided evidence for the presence of two neuroendocrine tumours. Uptake in the right neck corresponded to a chemodectoma, but there was no change in the clinical condition or fall in ACTH levels following surgical resection. Uptake in the left chest was assumed to relate to a bronchial carcinoid, but a tumour could not initially be localized on magnetic resonance imaging (MRI), spiral CT scanning or on selective venous sampling. A 1 cm bronchial carcinoid tumour was identified post-mortem which immunostained for ACTH. This case demonstrates that 111ln-octreotide scanning is a useful technique for identifying the source of ectopic ACTH production in difficult cases of Cushing's syndrome. Reliance should not be placed solely on standard imaging techniques to localize the tumour prior to surgery. Although rare, the possibility of a non-ACTH secreting neuroendocrine tumour should also be considered in patients with ectopic ACTH syndrome, who have positive 111In-octreotide scans.

Specific T cell deletion by transfected human monocytes expressing fas ligand and antigen.

The aim of our experiments was to determine whether deletion of antigen specific T helper cells could be accomplished by delivering the antigenic peptide to antigen presenting cells. Tetanus toxin peptide residues 830-843 was chosen for these experiments. Two mammalian expression vectors carrying the genes for human Fas ligand and a chimeric invariant chain-tetanus toxin peptide construct were designed. The T cell proliferative response to tetanus toxoid was inhibited when the antigen was presented by autologus monocytes transfected with Fas ligand. T cell mixture experiments using two syngeneic T cell lines specific either for tetanus toxoid or for pertussis toxin demonstrated that the killing effect elicited by the antigen pulsed/Fas ligand-transfected antigen presenting cells was antigen specific. Finally, we demonstrated that transient expression of antigen delivered by plasmid DNA can substitute for soluble antigen in the induction of antigen-specific T cell responses. Antigen presenting cells transfected with the vector carrying Fas ligand and the vector carrying the chimeric invariant chain-peptide antigen gene were shown to inhibit antigen specific T cell reactivity. This strategy may be useful for the induction of apoptosis in allopeptide reactive T cells driving chronic rejection.

A novel thyrotropin receptor mutation in an infant with severe thyrotoxicosis.

An infant girl was born at 37 weeks gestation and found to be clinically thyrotoxic at 9 months of age. Thyroid autoantibodies were negative, and thyroid function failed to normalize with medical treatment. The patient underwent a total thyroidectomy. DNA obtained from her thyroid gland and leukocytes was analyzed for thyrotropin receptor (TSHR) mutations using single strand conformation polymorphism and direct sequencing. A mobility shift of polymerase chain reaction (PCR)-amplified DNA was detected on single strand conformation polymorphism gel. Direct sequencing identified a novel point mutation in the fifth transmembrane domain of the TSH receptor at codon 597 (GTC to CTC), resulting in the amino acid substitution of leucine for valine. The mutation was heterozygous and germline, and was not identified in DNA from either of her parents. Expression of the V597L mutant is transiently transfected COS 7 cells displayed increased constitutive cyclic adenosine monophosphate (cAMP) production compared with the wild-type receptor. The mutant is expressed at very low levels on the surface of COS cells, and its response to TSH is marginal.

Mutation analysis of protein kinase A catalytic subunit in thyroid adenomas and pituitary tumours.

OBJECTIVE: The adenylyl cyclase system plays an important role in the control of both thyroid follicular and anterior pituitary cell function. Activating mutations affecting important pathway components such as the TSH receptor and Gsalpha occur in the majority of autonomously functioning thyroid nodules. Only a small proportion of other types of thyroid tumours, however, have been reported to harbour these mutations. Activating mutations of Gsalpha have been reported to occur in up to 40% of pituitary somatotroph adenomas. As the majority of cold thyroid nodules and pituitary tumours are unaffected by these mutations, we have investigated the possibility of activating mutations occurring in protein kinase A (PKA), which is another key component of the adenylyl cyclase pathway. DESIGN: Genomic DNA and cDNA were analysed for the presence of PKA Calpha mutations by allele-specific oligonucleotide hybridisation and single strand conformation polymorphism analysis. PATIENTS: A total of 171 tissue samples were investigated. These comprised 66 benign and 24 malignant thyroid neoplasms, 21 somatotroph adenomas, 35 non-functioning pituitary adenomas, 2 corticotroph adenomas, 1 malignant prolactinoma, and 22 normal pituitary tissue samples. RESULTS: No mutations of PKA Calpha were identified using either allele-specific oligonucleotide hybridisation or single strand conformation polymorphism analysis. CONCLUSIONS: It appears that PKA Calpha mutations at the codons investigated do not represent an oncogenetic mechanism in the development of thyroid and pituitary neoplasms.

Prevalence of Ras mutations in thyroid neoplasia.

OBJECTIVE: Mutations at codons 12, 13 or 61 of ras which result in constitutive activation occur frequently in human malignancies. There have been varied reports on their prevalence and hence their likely significance in the pathogenesis of primary thyroid neoplasia. To address this, we have examined a large series of benign and malignant thyroid tumours for ras mutations. DESIGN: Genomic DNA was analysed for the presence of mutations at codons 12, 13 and 61 of H-ras, K-ras and N-ras by allele-specific oligonucleotide hybridization. Direct DNA sequencing was used to confirm the mutations. PATIENTS: A total of 90 samples with benign (66) and malignant (24) thyroid disease were investigated. RESULTS: A total of 14/90 (15.5%) samples had a ras mutation. All mutations were at codon 61 of either N-ras or K-ras. The positive cases were 1/25 (4%) nodular goitre, 7/38 (18%) follicular adenoma, 4/9 (44%) follicular carcinoma, 1/1 anaplastic carcinoma, 1/1 follicular variant of papillary carcinoma, and 1 metastatic follicular carcinoma in which the primary tumour had the same mutation. CONCLUSIONS: Our data demonstrate a relatively low overall prevalence of ras mutations in thyroid neoplasia, with a predominance in follicular neoplasms. Their presence in follicular adenomas suggests that they may have an early aetiological role in the development of thyroid neoplasia.

Wild-type p53 epitope naturally processed and presented by an HLA-B haplotype on human breast carcinoma cells.

To broaden the clinical applicability of peptide-based immunotherapy in breast cancer, there is a need to identify further tumor-associated peptide epitopes that are specific for HLA alleles, in addition to HLA-A2. The HLA-B44 haplotype is one of the most common HLA-B haplotypes, occurring in 10-20% of the population. We performed the structural characterization of HLA class I-bound self-peptides presented by a human breast cancer cell line with a HLA-A68, A32, B40, B44 haplotype, to identify potential tumor-specific antigens. Of 13 sequenced peptides, 1 peptide had the HLA-A68 peptide binding motif and 12 peptides had the HLA-B40, B44 peptide binding motif. One of the latter peptides, FEVRVCACPG, shared 100% homology to residues 270-279 of wild-type P53 protein. Our study, thus, provides direct evidence for the natural processing and presentation of p53 epitope 270-279 by HLA-B40, B44-bearing human breast tumor cells. Epitopes spanning this region of P53 may have potential use for immunotherapy in patients expressing HLA-A2 and -B44 supertypes.

Indirect allorecognition in solid organ transplantation.

Recipient T cell recognition of donor major histocompatibility complex (MHC) alloantigens plays a central role in both acute and chronic rejection of human organ allografts. Two different pathways of T cell recognition of donor MHC alloantigens have been described. The direct pathway involves T cell recognition of intact MHC molecules expressed by donor antigen-presenting cells (APCs). The second, or indirect pathway, operates via T helper cell recognition of peptides derived from the processing and presentation of allogeneic MHC molecules on self-APCs. At the onset of primary acute rejection, recipient CD4+ T cell responses to donor HLA-DR alloantigens are limited to a single dominant determinant present on one of the disparate alloantigens and restricted by one of the responder's HLA-DR molecules. In allograft recipients with recurring episodes of rejection, and/or at the onset of chronic rejection, recipient T cell reactivity may spread to other epitopes within the allogeneic MHC molecule, as well as to other alloantigens expressed by graft tissue. Both quantitative and qualitative alterations in T cell allopeptide reactivity are associated with increased risk of cellular and/or humoral rejection. These studies provide a basis for the design of new therapeutic strategies and for immunologic monitoring of transplant recipients.

Ligation of HLA class I molecules on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation.

The development of transplant atherosclerosis, a manifestation of chronic rejection, is the major obstacle to long-term survival of cardiac and renal allografts. The incidence of transplant atherosclerosis is increased in transplant recipients producing antidonor HLA antibodies following transplantation, suggesting that anti-HLA antibodies play a role in the pathogenesis of the disease. We have postulated that anti-HLA antibodies mediate the development of transplant atherosclerosis by binding to class I molecules on the endothelium and smooth muscle of the graft and transducing signals which stimulate cell proliferation. In this report we demonstrate that anti-HLA class I antibodies transduce signals in smooth muscle cells stimulating increased tyrosine phosphorylation of intracellular proteins and up-regulation of fibroblast growth factor (FGF) receptors. Antibody binding to class I molecules on smooth muscle cells is also accompanied by increased responsiveness to basic FGF and augmented cell proliferation. These findings may explain the increased occurrence of transplant atherosclerosis in recipients producing anti-donor HLA antibodies.

Intramolecular and intermolecular spreading during the course of organ allograft rejection.

There are two distinct pathways by which T cells may recognize MHC alloantigens. The direct pathway involves T-cell recognition of intact MHC molecules expressed by donor antigen-presenting cells (APCs). The second, or indirect, pathway describes T-cell recognition of peptides derived from the processing and presentation of allogeneic MHC molecules on self APCs. Recent data demonstrates that indirect recognition plays a central role in both acute and chronic rejection of human organ allografts. Our studies have shown that, at the onset of primary acute rejection, recipient T-cell responses to donor HLA-DR alloantigens are limited to a single dominant determinant present on one of the disparate alloantigens and restricted by one of the responder's HLA-DR molecules. In allograft recipients with recurring episodes of rejection, and/or at the onset of chronic rejection, recipient T-cell reactivity may spread to other epitopes within the allogeneic MHC molecule as well as to other alloantigens expressed by graft tissue. Both quantitative and qualitative alterations in T-cell allopeptide reactivity are associated with increased risk of cellular and/or humoral rejection. These studies provide a basis for the design of new therapeutic strategies and for immunologic monitoring of transplant recipients.