X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

SARS-CoV-2 Infection and Antibody Seroprevalence among UK Healthcare Professionals Working with Cancer Patients during the First Wave of the COVID-19 Pandemic.

AIMS: The proportion of UK oncology healthcare professionals (HCPs) infected with SARS-CoV-2 during the COVID-19 pandemic's first wave is unknown. The primary aim of this study was to determine the SARS-CoV-2 infection and seroprevalence rates among HCPs. MATERIALS AND METHODS: Patient-facing oncology HCPs working at three large UK hospitals during the COVID-19 pandemic's first wave underwent polymerase chain reaction (PCR) and antibody testing [Luminex and point-of-care (POC) tests] on two occasions 28 days apart (June-July 2020). RESULTS: In total, 434 HCPs were recruited: nurses (58.3%), doctors (21.2%), radiographers (10.4%), administrators (10.1%); 26.3% reported prior symptoms suggestive of SARS-CoV-2. All participants were PCR negative during the study, but 18.4% were Luminex seropositive on day 1, of whom 42.5% were POC seropositive. Nurses had the highest seropositive prevalence trend (21.3%, P = 0.2). Thirty-eight per cent of seropositive HCPs reported previous SARS-CoV-2 symptoms: 1.9 times higher odds than seronegative HCPs (P = 0.01). Of 400 participants retested on day 28, 13.3% were Luminex seropositive (92.5% previously, 7.5% newly). Thirty-two per cent of initially seropositive HCPs were seronegative on day 28. CONCLUSION: In this large cohort of PCR-negative patient-facing oncology HCPs, almost one in five were SARS-CoV-2 antibody positive at the start of the pandemic's first wave. Our findings that one in three seropositive HCPs retested 28 days later became seronegative support regular SARS-CoV-2 PCR and antibody testing until widespread immunity is achieved by effective vaccination.

Detection of breastmilk antibodies targeting SARS-CoV-2 nucleocapsid, spike and receptor-binding-domain antigens.

A 40-year-old female was found to have strongly neutralizing SARS-CoV-2 breastmilk IgA and IgG antibodies reactive against multiple SARS-CoV-2 antigens at 2.5 months after documented infection with SARS-CoV-2. At 6.5 months following the infection, she remained positive for breastmilk and serum SARS-CoV-2 neutralizing antibodies. Holder breast milk pasteurization did not diminish SARS-CoV-2 antibody titres but it reduced its neutralizing capacity, while serum heat inactivation had no negative effect on SARS-CoV-2 serum antibody levels and neutralizing capacity. Current data on SARS-CoV-2 and breastmilk are reviewed.

Neutralising antibodies in Spike mediated SARS-CoV-2 adaptation.

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against ΔH69/ΔV70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

High incidence of anti-cytokine autoantibodies in dogs with immune diseases suggests important immuno-regulatory functions.

Autoantibodies against cytokines have been associated with immunodeficiency, susceptibility to infectious diseases, autoimmunity and inflammation in humans, but have not yet been investigated in the Veterinary field so far. The aim of the current study was to determine the presence of anti-cytokine autoantibodies in canines suffering from various conditions including recurrent infections, autoimmune diseases and cancer in comparison to healthy controls. This is the first report of the presence of autoantibodies against cytokines in dogs. A total of 101 serum samples (51 patients and 50 clinically healthy dogs) from the state of Mexico and surroundings were analysed using a multiplex bead-based flow cytometry assay. Results show significant levels of various anti-cytokine autoantibodies in diseased dogs but not in healthy controls. In addition we show distinct associations of various disease types to the specificity of anti-cytokine autoantibodies and to response complexities. Apart from the direct functional/causal implication of anti-cytokine auto-antibodies on disease processes, this findings point to the possibility to use anti-cytokine response patterns as diagnostic tools.

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.

In a novel form of IFN-gamma receptor 1 deficiency, cell surface receptors fail to bind IFN-gamma.

Complete IFN-gamma receptor ligand-binding chain (IFNgammaR1) deficiency is a life-threatening autosomal recessive immune disorder. Affected children invariably die of mycobacterial infection, unless bone marrow transplantation is undertaken. Pathogenic IFNGR1 mutations identified to date include nonsense and splice mutations and frameshift deletions and insertions. All result in a premature stop codon upstream from the segment encoding the transmembrane domain, precluding cell surface expression of the receptors. We report herein two sporadic and two familial cases of a novel form of complete IFNgammaR1 deficiency in which normal numbers of receptors are detected at the cell surface. Two in-frame deletions and two missense IFNGR1 mutations were identified in the segment encoding the extracellular ligand-binding domain of the receptor. Eight independent IFNgammaR1-specific mAb's, including seven blocking antibodies, gave recognition patterns that differed between patients, suggesting that different epitopes were altered by the mutations. No specific binding of (125)I-IFN-gamma to cells was observed in any patient, however, and the cells failed to respond to IFN-gamma. The mutations therefore cause complete IFNgammaR1 deficiency by disrupting the IFN-gamma-binding site without affecting surface expression. The detection of surface IFNgammaR1 molecules by specific antibodies, including blocking antibodies, does not exclude a diagnosis of complete IFNgammaR1 deficiency.

IL-12 and IFN-gamma in host defense against mycobacteria and salmonella in mice and men.

The development of gene-knockout mice and the identification of gene-deficient humans have improved our understanding of the role of IL-12 and IFN-gamma in host defense. Comparison of experimental and natural infections has shown that animals and humans genetically deficient in immunity mediated by IL-12 or IFN-gamma are highly susceptible to mycobacteria and salmonella. Impaired secretion of, or response to, IFN-gamma is the common pathogenic mechanism that accounts for impaired granuloma formation and uncontrolled growth of bacteria within macrophages. The axis formed between IL-12 and IFN-gamma is essential for protective immunity against mycobacteria and salmonella in mice and men.

Mendelian susceptibility to mycobacterial infection in man.

Selective susceptibility to poorly pathogenic mycobacteria, such as bacille Calmette-Guérin vaccine and environmental non-tuberculous mycobacteria, has long been suspected to be a mendelian disorder but its molecular basis has remained elusive. Recently, recessive mutations in the interferon-gamma-receptor receptor ligand-binding chain, interferon-gamma-receptor signalling chain, IL-12 p40 subunit and IL-12-receptor beta 1 chain genes have been identified in a number of patients with disseminated mycobacterial infection. Although genetically distinct, these conditions are immunologically related and highlight the essential role of interferon-gamma-mediated immunity in the control of mycobacteria in man.

Inherited disorders of IL-12- and IFNgamma-mediated immunity: a molecular genetics update.

In the last 6 years, considerable advances have been made in the molecular analysis of a rare clinical syndrome: Mendelian susceptibility to mycobacterial disease (MSMD). Infection with poorly virulent environmental non-tuberculous mycobacteria (NTM) or vaccination with bacillus Calmette-Guerin (BCG) may cause disseminating and even fatal disease in individuals suffering from this syndrome. Mutations in five genes (IFNGR1, IFNGR2, STAT1, IL12B and IL12RB1) have been shown to be responsible for MSMD and further allelic heterogeneity accounts for the existence of nine distinct inherited disorders. All of these disorders are caused by impaired IFNgamma-mediated immunity. These results have important medical and biological implications. In this report, we update the disease-causing mutations reported in the literature.

The MHC class II-restricted T cell response of C57BL/6 mice to human C-reactive protein: homology to self and the selection of T cell epitopes and T cell receptors.

The T cell response of C57BL/6 mice to human C-reactive protein (hCRP), an inducible acute phase protein, was analysed. Two I-A(b)-restricted epitopes at positions 79 95 (epitope A) and 87-102 (epitope B) were identified using a panel of CD4+ T cell clones. Human C-reactive protein shares considerable homology with mouse C-reactive protein and mouse serum amyloid P component. Interestingly, the two epitopes map to the region of lowest homology between human CRP and its mouse homologues. Human CRP-specific T cell clones express a restricted T cell receptor (TCR) repertoire, both with regard to usage of TCR germline gene segments (V alpha, J alpha, V beta, J beta) and certain TCR alpha beta combinations. Therefore, epitope-A specific clones preferentially use TCR V beta8.3 and V alpha3 J alpha15 V beta8.3-J beta2.3 and epitope-B specific clones use V beta2 and V alpha1-J alpha24/30-V beta2. This bias is even more pronounced when TCR usage is correlated with epitope fine specificity. A role for homology of hCRP to self components in selecting these particular T cell epitopes and TCR is discussed.

Genetic heterogeneity of Mendelian susceptibility to mycobacterial infection.

Mendelian susceptibility to poorly virulent mycobacterial species, such as bacillus Calmette-Guérin (BCG) and environmental nontuberculous mycobacteria (NTM), is a phenotypically heterogeneous syndrome. It has therefore long been suspected to be genetically heterogeneous. In the past 5 years, this prediction has been confirmed and different types of mutations (dominant or recessive, nonfunctional or hypofunctional) in four genes (IFNGR1, IFNGR2, IL12B, IL12RB1) have revealed both allelic and nonallelic heterogeneity. The eight disorders resulting from these mutations are genetically different but immunologically related, as impaired IFN-gamma-mediated immunity is the common pathogenic mechanism accounting for mycobacterial infection in all patients. The severity of the phenotype depends on the genotype. Complete IFN-gammaR1 and IFN-gammaR2 deficiencies predispose patients to a more severe clinical course than partial IFN-gammaR1 and IFN-gammaR2 deficiencies and complete IL-12 p40 and IL-12Rbeta1 deficiencies.

Mycobacterial diseases in primary immunodeficiencies.

Primary immunodeficiency diseases comprise over 100 conditions, each associated with a variety of viral, bacterial, fungal and protozoan infections. M. tuberculosis and less virulent mycobacteria, such as bacille Calmette-Guérin vaccines and environmental non-tuberculous mycobacteria, may cause severe disease in patients with primary immunodeficiency diseases. However, no previous review has dealt with the issue of which primary immunodeficiency diseases predispose affected individuals to mycobacterial disease. This information is very useful, not only increasing our understanding of human immunity to mycobacteria, but also for the diagnostic investigation of patients with mycobacteriosis. We review here the medical literature on cases of mycobacterial disease in patients with primary immunodeficiency diseases.

Genetically determined susceptibility to mycobacterial infection.

Individuals with impaired cell mediated immunity exhibit increased susceptibility to infections caused by poorly pathogenic mycobacteria (non-tuberculous mycobacteria and BCG), as well as salmonella species. However, these infections may also occur in a disseminated, fatal form, sometimes with a familial distribution, in the absence of any recognised primary or secondary immunodeficiency. Genetic analysis of affected families has defined mutations in seven different genes participating in the interleukin 12 (IL12) dependent, high output interferon gamma (IFNgamma) pathway. The first category of defect is mutations in the IFNgammaR1 or R2 genes, resulting in defective expression or function of the IFNgamma receptor. The second category of mutations abrogates the cell surface expression IL12Rbeta1gene, resulting in the inability to respond to IL12. The third category of defect is the inability to produce IL12, due to deletion within the gene coding for the inducible chain of IL12 (IL12-p40). Patients with X-linked recessive mutations of the gene encoding the NFkappaB essential modulator may also develop mycobacterial infections, although they usually have a more complex phenotype and are susceptible to a broad spectrum of pathogens. Mutations of the gene encoding the signal transducing molecule STAT1, which impairs the ability to respond to IFNgamma, and mutations of the gene encoding TYK2 (which is associated with a failure to respond to IL12), are both rare genetic defects predisposing to mycobacterial infections. This review summarises the clinical spectrum seen in this group of patients and indicates a strategy for the identification of putative genetic defects in the type-1 cytokine pathway.

The effect of sirolimus therapy on vaccine responses in transplant recipients.

Different immunosuppressant regimens vary in their effects on antibody responses to vaccination. The combination of prednisolone and azathioprine has only a minor effect, whereas the addition of ciclosporin attenuates protective antibody responses to influenza vaccination. The effect of sirolimus, a new immunosuppressant, on vaccine responses has been little studied. Thirty-two hepatic or renal transplant patients randomized to calcineurin inhibitor-based or sirolimus-based immunosuppression were vaccinated against influenza and pneumococcus. Following tri-valent influenza vaccination, a similar rise in antibody titer occurred in sirolimus and calcineurin inhibitor (CNI) treated patients, though sirolimus treated patients developed a 'protective' titer to more influenza antigens. The pneumococcal polysaccharide vaccine was equally effective in both groups. Hence, vaccination guidelines in place for CNI treated patients are likely to be appropriate for transplant recipients maintained on sirolimus.

Testing for electrotransfection parameters by use of the fluorescent dye lucifer yellow CH.

A nonradioactive and rapid method to systematically optimize conditions for electrotransfection is described here for a critical parameter, the initial voltage. This technique utilizes the electric field-dependent transfer of the fluorescent compound Lucifer Yellow CH into cells. Dye uptake can be followed and quantified by fluorescence microscopy for individual cells or in sum by fluorescence spectroscopy. Electrotransfection conditions for maximal dye and DNA uptake correspond with each other. Cotransfection of Lucifer Yellow CH and DNA coding for the indicator gene chloramphenicol acetyltransferase followed by fluorescence-activated cell sorting demonstrates that the cell population that takes up the fluorescent compound also expresses the indicator gene.