Enrichment of Immune Dysregulation Disorders in Adult Patients with Human Inborn Errors of Immunity.

Human inborn errors of immunity (IEI) comprise a group of diseases resulting from molecular variants that compromise innate and adaptive immunity. Clinical features of IEI patients are dominated by susceptibility to a spectrum of infectious diseases, as well as autoimmune, autoinflammatory, allergic, and malignant phenotypes that usually appear in childhood, which is when the diagnosis is typically made. However, some IEI patients are identified in adulthood due to symptomatic delay of the disease or other reasons that prevent the request for a molecular study. The application of next-generation sequencing (NGS) as a diagnostic technique has given rise to an ever-increasing identification of IEI-monogenic causes, thus improving the diagnostic yield and facilitating the possibility of personalized treatment. This work was a retrospective study of 173 adults with IEI suspicion that were sequenced between 2005 and 2023. Sanger, targeted gene-panel, and whole exome sequencing were used for molecular diagnosis. Disease-causing variants were identified in 44 of 173 (25.43%) patients. The clinical phenotype of these 44 patients was mostly related to infection susceptibility (63.64%). An enrichment of immune dysregulation diseases was found when cohorts with molecular diagnosis were compared to those without. Immune dysregulation disorders, group 4 from the International Union of Immunological Societies Expert Committee (IUIS), were the most prevalent among these adult patients. Immune dysregulation as a new item in the Jeffrey Model Foundation warning signs for adults significantly increases the sensitivity for the identification of patients with an IEI-producing molecular defect.

Rationale for combined therapies in severe-to-critical COVID-19 patients.

An unprecedented global social and economic impact as well as a significant number of fatalities have been brought on by the coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acute SARS-CoV-2 infection can, in certain situations, cause immunological abnormalities, leading to an anomalous innate and adaptive immune response. While most patients only experience mild symptoms and recover without the need for mechanical ventilation, a substantial percentage of those who are affected develop severe respiratory illness, which can be fatal. The absence of effective therapies when disease progresses to a very severe condition coupled with the incomplete understanding of COVID-19's pathogenesis triggers the need to develop innovative therapeutic approaches for patients at high risk of mortality. As a result, we investigate the potential contribution of promising combinatorial cell therapy to prevent death in critical patients.

Humans with inherited MyD88 and IRAK-4 deficiencies are predisposed to hypoxemic COVID-19 pneumonia.

X-linked recessive deficiency of TLR7, a MyD88- and IRAK-4-dependent endosomal ssRNA sensor, impairs SARS-CoV-2 recognition and type I IFN production in plasmacytoid dendritic cells (pDCs), thereby underlying hypoxemic COVID-19 pneumonia with high penetrance. We report 22 unvaccinated patients with autosomal recessive MyD88 or IRAK-4 deficiency infected with SARS-CoV-2 (mean age: 10.9 yr; 2 mo to 24 yr), originating from 17 kindreds from eight countries on three continents. 16 patients were hospitalized: six with moderate, four with severe, and six with critical pneumonia, one of whom died. The risk of hypoxemic pneumonia increased with age. The risk of invasive mechanical ventilation was also much greater than in age-matched controls from the general population (OR: 74.7, 95% CI: 26.8-207.8, P < 0.001). The patients' susceptibility to SARS-CoV-2 can be attributed to impaired TLR7-dependent type I IFN production by pDCs, which do not sense SARS-CoV-2 correctly. Patients with inherited MyD88 or IRAK-4 deficiency were long thought to be selectively vulnerable to pyogenic bacteria, but also have a high risk of hypoxemic COVID-19 pneumonia.

The age again in the eye of the COVID-19 storm: evidence-based decision making.

BACKGROUND: One hundred fifty million contagions, more than 3 million deaths and little more than 1 year of COVID-19 have changed our lives and our health management systems forever. Ageing is known to be one of the significant determinants for COVID-19 severity. Two main reasons underlie this: immunosenescence and age correlation with main COVID-19 comorbidities such as hypertension or dyslipidaemia. This study has two aims. The first is to obtain cut-off points for laboratory parameters that can help us in clinical decision-making. The second one is to analyse the effect of pandemic lockdown on epidemiological, clinical, and laboratory parameters concerning the severity of the COVID-19. For these purposes, 257 of SARSCoV2 inpatients during pandemic confinement were included in this study. Moreover, 584 case records from a previously analysed series, were compared with the present study data. RESULTS: Concerning the characteristics of lockdown series, mild cases accounted for 14.4, 54.1% were moderate and 31.5%, severe. There were 32.5% of home contagions, 26.3% community transmissions, 22.5% nursing home contagions, and 8.8% corresponding to frontline worker contagions regarding epidemiological features. Age > 60 and male sex are hereby confirmed as severity determinants. Equally, higher severity was significantly associated with higher IL6, CRP, ferritin, LDH, and leukocyte counts, and a lower percentage of lymphocyte, CD4 and CD8 count. Comparing this cohort with a previous 584-cases series, mild cases were less than those analysed in the first moment of the pandemic and dyslipidaemia became more frequent than before. IL-6, CRP and LDH values above 69 pg/mL, 97 mg/L and 328 U/L respectively, as well as a CD4 T-cell count below 535 cells/µL, were the best cut-offs predicting severity since these parameters offered reliable areas under the curve. CONCLUSION: Age and sex together with selected laboratory parameters on admission can help us predict COVID-19 severity and, therefore, make clinical and resource management decisions. Demographic features associated with lockdown might affect the homogeneity of the data and the robustness of the results.

COVID-19: age, Interleukin-6, C-reactive protein, and lymphocytes as key clues from a multicentre retrospective study.

BACKGROUND: The SARS-CoV-2 infection has widely spread to become the greatest public health challenge to date, the COVID-19 pandemic. Different fatality rates among countries are probably due to non-standardized records being carried out by local health authorities. The Spanish case-fatality rate is 11.22%, far higher than those reported in Asia or by other European countries. A multicentre retrospective study of demographic, clinical, laboratory and immunological features of 584 Spanish COVID-19 hospitalized patients and their outcomes was performed. The use of renin-angiotensin system blockers was also analysed as a risk factor. RESULTS: In this study, 27.4% of cases presented a mild course, 42.1% a moderate one and for 30.5% of cases, the course was severe. Ages ranged from 18 to 98 (average 63). Almost 60 % (59.8%) of patients were male. Interleukin 6 was higher as severity increased. On the other hand, CD8 lymphocyte count was significantly lower as severity grew and subpopulations CD4, CD8, CD19, and NK showed concordant lowering trends. Severity-related natural killer percent descents were evidenced just within aged cases. A significant severity-related decrease of CD4 lymphocytes was found in males. The use of angiotensin-converting enzyme inhibitors was associated with a better prognosis. The angiotensin II receptor blocker use was associated with a more severe course. CONCLUSIONS: Age and age-related comorbidities, such as dyslipidaemia, hypertension or diabetes, determined more frequent severe forms of the disease in this study than in previous literature cohorts. Our cases are older than those so far reported and the clinical course of the disease is found to be impaired by age. Immunosenescence might be therefore a suitable explanation for the hampering of immune system effectors. The adaptive immunity would become exhausted and a strong but ineffective and almost deleterious innate response would account for COVID-19 severity. Angiotensin-converting enzyme inhibitors used by hypertensive patients have a protective effect in regards to COVID-19 severity in our series. Conversely, patients on angiotensin II receptor blockers showed a severer disease.

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Evaluation of different competitive immunoassays for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry.

Haptens (i.e. biomolecules which molecular weight is lower than 10 kDa) determination by inductively coupled plasma mass spectrometry (ICP-MS) is usually performed by means of competitive immunoassays. In these immunoassays, analyte quantification is indirectly carried out using two different tracer species (i.e. antibodies or antigen-protein conjugates). However, the benefits (and drawbacks) derived from using a given tracer species have not been systematically investigated so far. The goal of this work is to evaluate the influence of the tracer species employed in competitive immunoassays on the analytical figures of merit for aflatoxin M1 (AFM1) determination in milk samples. To this end, three different strategies have been developed and evaluated, namely: (i) antibody binding inhibition assay (ABIA); (ii) capture inhibition assay (CIA); and (iii) capture bridge inhibition assay (CBIA). Experimental results show that the use of the antibody as tracer species (as in the ABIA approach) affords better analytical figures of merit for AFM1 determination than using the antigen-protein conjugate as the tracer one (as in the CIA and CBIA strategies). The limit of detection afforded by ABIA strategy (i.e. 0.1 ng kg-1) for AFM1 determination was 1000-fold and 50-fold lower regarding the CIA and CBIA strategies, respectively. In the case of the ABIA approach, the characteristics of the metal nanoparticle label employed to detect the tracer species is critical on the analytical figures of merit. However, when the hapten-protein conjugates are used as tracer species, immunocomplex formation is severely hampered by steric effects caused by the protein moiety and, consequently, the characteristics of the metal nanoparticle label is not critical in the immunoassay performance. The different immunoassay strategies were successfully validated for AFM1 determination in milk samples using a certified reference material of whole milk powder (ERM-BD283) according to European Conformity guidelines for analytical methods of food contaminants and mycotoxins. Compared to ELISA, the immunoassay developed for AFM1 determination in milk samples improve limits of detection up to 10-fold.

Bacterial vaccines in chronic obstructive pulmonary disease: effects on clinical outcomes and cytokine levels.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality and morbidity worldwide. Exacerbation episodes impair lung function leading to disease progression. Levels of inflammation markers correlate with disease severity. Bacterial immunomodulators have shown a beneficial effect in COPD, improving symptoms and reducing the rate of exacerbations. This is an observational prospective study on 30 patients diagnosed with bronchiectasis and COPD, who received bacterial autogenous vaccine for 12 months. The rate of exacerbation, severity of symptoms and lung function were studied at baseline and after treatment. In addition, plasma levels CRP, IL6, IL8, and TNFα were measured. After treatment we found a reduction in mean acute respiratory infections and signs of lung disease. Acute phase proteins IL6 and CRP increased in blood and IL8 decreased. These changes may be related to the repeated injection of inactivated bacteria. Given the implication of these factors in the pathogenesis of COPD, particularly the production of IL8, the causes and consequences of cytokine modulation by bacterial vaccines should be investigated. Vaccination with autogenous vaccines for 1 year can produce a significant clinical improvement in COPD patients, reducing the frequency of exacerbations associated to changes in the profile of markers of inflammation.

Allergenicity of casein containing chalk in milk allergic schoolchildren.

BACKGROUND: Nondietary exposure to milk proteins may be a risk for children who do not outgrow milk allergy by school age. OBJECTIVE: To study the allergenicity of casein containing chalk. METHODS: A 6-year-old, milk allergic child developed asthma and rhinoconjunctivitis while in school. The suspected cause was dust-free chalk containing casein. To study the relationship of dust-free chalk containing casein with asthma and rhinoconjunctivitis, 13 additional milk allergic patients were studied: 3 school-aged children, 8 preschool-aged infants, and 2 children with outgrown milk allergy. Skin tests and/or specific IgE with chalk and casein were performed. A chalk use test was performed in older children. Milk allergens contained in chalk were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and IgE inhibition experiments. RESULTS: All school-aged, milk allergic children were exposed to chalk and reported symptoms attributed to chalk exposure. The skin test result to chalk was positive in 5 of 12 cases, and the specific IgE test result was positive in all 12 study participants in which it was performed. Casein strongly inhibited the binding of IgE to chalk. Chalk sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins with molecular weight similar to caseins. Immunoblot demonstrated strong binding of IgE to chalk in a blurred pattern and a band at 30 kDa, inhibited by casein. The chalk challenge test result was positive in 2 school-age children who had a positive skin test result to chalk. Their symptoms improved after avoidance of chalk in the school. In 2 other cases in which the challenge test result was negative, chalk was reintroduced without problems. CONCLUSION: Inhalation of chalk dust containing casein can induce asthma symptoms in milk allergic patients. Hidden and nondietary sources of exposure should always be considered in food allergic patients.

Acute allergic reaction to an iron compound in a milk-allergic patient.

A child allergic to cow's milk developed a mild systemic allergic reaction after the first dose of Ferplex 40 (iron proteinsuccinylate). Skin tests and in vitro studies were performed in the child, in three cow's milk-allergic controls and in a non-allergic control. Milk, casein and iron proteinsuccinylate (Ferplex 40) were used for skin tests, specific immunoglobulin E (IgE) determination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and enzyme allergo sorbent test (EAST) inhibition. A review of the drug information sheet and contact with the manufacturer were also performed. Although proteinsuccinylate is indeed a succinylated casein (each dose containing about 575 mg of casein) there was no indication of the milk protein content in the prescribing information provided by the manufacturer. Skin tests and specific IgE were positive in the case and in all allergic controls, except for EAST to iron proteinsuccinylate in one control. In EAST, iron proteinsuccinylate in solid phase was 100% inhibited by casein and casein in solid phase was inhibited 74% by iron proteinsuccinylate. SDS-PAGE of iron proteinsuccinylate showed a broad 46 kDa band and a blur of aggregated material. On immunoblot, the patient's IgE reacted to this heavily aggregated material and in the native casein extract recognized a 35-kDa band. The allergenicity of succinylated casein (proteinsuccinylate) among milk-allergic children is demonstrated. The protein source used in drug-protein conjugates should always be indicated by the manufacturer (as it should be in foods) to avoid potential risks to allergic patients.

Nonviral therapeutic cell vaccine mediates potent antitumor effects.

Therapeutic vaccination of mice bearing melanoma tumors with our genetically modified tumor cells, via DOTAP/GM-CSF lipoplexes, results in >85% tumor growth inhibition. These fresh transfected cells (irradiated, frozen and thawed) are able to produce high amounts of GM-CSF transgene (>200 ng/10(6) cells/24 h). After vaccination, significant increases (>eight-fold) in specific antitumor membrane protein IgG1 and IgG2a are obtained only in groups vaccinated with GM-CSF-producing cells, where also the highest rates of tumor inhibition, and significantly delayed mice death (P<0.05), are observed. The antitumor response obtained is long-lasting in survivors (GM-CSF-group) from 6 months after the first tumor challenge, and a full 100% of mice survived to a second tumor challenge. All these results suggest that antitumor cell vaccines engineered by nonviral procedures are suitable for use as therapeutic vaccines with potential clinical applications.

Occupational asthma to carmine in a butcher.

UNLABELLED: Hypersensitivity to carmine (E120) has been identified as a cause of food intolerance and occupational asthma. We present a case of occupational asthma following exposure to carmine in a manufacturer of sausages and review the literature. CASE REPORT: A 42-year-old non-atopic male presented with a 5-year history of rhinoconjunctivitis and asthma on occupational exposure to food additive dusts. Symptoms increased after work. The patient had been exposed for more than 20 years. METHODS: Skin prick tests were performed with a battery of common inhalant allergens and spices. Cochineal, carmine lake and additive mixes used by the patient were extracted and subsequently used for skin prick test, bronchial provocation and in vitro measurements (specific IgE, Western blot and chromatographic fractionation). RESULTS: Prick tests were positive to carmine and carmine-containing additives; carmine-specific IgE and bronchial challenge tests were also positive (PC20 = 0.0004 mg/ml and 1.6 kU/l). Western blot showed IgE binding to bands of about 30 kDa on cochineal extract and a diffuse pattern at 40-97 kDa on carmine. This result was confirmed by gel filtration chromatography and dot blot. Carmine completely inhibited IgE binding to cochineal extract. DISCUSSION: Carmine is a potential sensitizer in an occupational setting: 18 cases of occupational asthma have been described to date. Carmine allergens are poorly defined; in general, proteins from cochineal not removed by the extraction process are considered as the main allergens in carmine. Our results are consistent with this, but show that these proteins may be subject to chemical modification.

Complete tumor prevention by engineered tumor cell vaccines employing nonviral vectors.

We report that 100% mice survival after tumor challenge is achieved with cytokine-engineered cells employing nonviral lipoplexes and without using viral vectors. We describe this effect with cytokine-secreting tumor cell vaccines, based on cell clones or fresh transfected cells. Tumor cells were transfected with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 plasmids employing the cationic lipid DOTAP, were irradiated (150 Gy) and kept frozen until use. The transfection efficacy was analyzed by qRT-PCR and flow cytometry. Vaccination induced potent antitumor rejection, resulting in 100% mice survival. Furthermore, the antitumor immunity was long lasting, since a two-fold survival delay was observed in mice after tumor rechallenge (6 months later). While cell clones secreting GM-CSF were the most effective in wild-type tumor cell rejection, little or no effect was observed with clones secreting IL-4. We found similar antitumor efficacy employing fresh transfected cells by nonviral procedures, demonstrating that cells genetically modified by nonviral vectors (both clones and fresh transfected cells) are a safe and efficient tool for antitumor vaccines. These vaccines allow us to achieve the highest antitumor efficacy based on nonviral gene therapy techniques. In addition, the vaccination success with fresh transfected cells simplifies the procedure and provides new insights into the clinical application of nonviral gene therapy procedures.