Enteropathies of newborns, symptoms, treatment. Immunodeficiencies caused by impaired cellular immunity
Abstract
This disease is characterized by the onset of primary immunodeficiency, which expresses itself as autoimmune multisystem failure, often clinically manifests during the first year of life; there are only about 150 cases in the world described by now. IPEX syndrome is caused by FOXP3 gene defect, which is a transcription factor that affects the activity of regulatory T-cells responsible for the maintenance of aytotolerance. There are around 70 pathogenic mutations in this gene described so far. Most patients with IPEX-syndrome have a clinical manifestations of the disease in the early neonatal period or during the first 3-4 months of life. For this disease the following clinical triad of manifestations is typical: Autoimmune enteropathy (100%), diabetes mellitus (70%), skin lesions (65%), as in the syndrome structure includes severe developmental delay (50%), thyroid disease ( 30%), recurrent infections (20%), rare autoimmune cytopenia (Coombs-positive hemolytic anemia), pneumonia, nephritis, hepatitis, artrit, myositis, alopecia. However, some cases of later manifestations were described (in patients of more than 1 year of age) when patients did not show all clinical and laboratory symptoms typical for severe forms of the disease. Due to the severity of the disease and the high mortality in this group of patients, it is very important to diagnose it early and start therapy timely. The article describes a clinical case of permanent neonatal diabetes mellitus in the structure of IPEX syndrome.
IPEX syndrome (Immunodeficiency, Polyendocrinopathy, Enteropathy, X-linked Syndrome). Synonyms: XLAAD (X-linked Autoimmunity-Allergic Dysregulation Syndrome) - rare disease; About 150 cases have been described in the world. According to some foreign sources, the prevalence of IPEX syndrome among patients with permanent neonatal diabetes mellitus is about 4%. This syndrome is characterized by the occurrence of primary immunodeficiency, which manifests itself as autoimmune multiple organ damage and most often manifests clinically in the first year of life. In 1982, Powel et al. first described a family in which 19 males had an X-linked disease manifested by diarrhea and polyendocrinopathy, including insulin-dependent diabetes mellitus. Later in 2000, Catila et al. identified a mutation in the gene encoding the C-terminal DNA binding domain (FKN) in two various patients male with similar clinical picture. In 2000-2001 Bennett et al. and Wildin et al. independently confirmed that IPEX syndrome is based on mutations in the FOXP3 gene. Currently, about 70 pathogenic mutations of this gene have been described. The FOXP3 gene is a transcription factor that influences the activity of regulatory T cells responsible for maintaining self-tolerance. According to data published by Barzaghi et al. in 2012, the main mechanism of autoimmune organ damage in IPEX syndrome is considered to be a dysfunction of regulatory T cells. Thus, IPEX syndrome is characterized by the development of severe immunodeficiency, which can lead to septic complications and often death. Currently, about 300 genes are known that lead to the development primary immunodeficiencies(PID). Previously, it was believed that these diseases were very rare, but recent studies indicate their significant prevalence. It is extremely important for pediatricians to be alert to the possible presence of PID, especially in cases of severe infections in combination with autoimmune diseases in children. In most patients with IPEX syndrome, clinical manifestations of the disease begin in the early neonatal period or during the first 3-4 months of life. A clinical triad of manifestations is typical for this pathology: autoimmune enteropathy (100%), diabetes mellitus (70%), skin lesions (65%), the structure of the syndrome also includes severe developmental delay (50%), damage to the thyroid gland (30%), recurrent infections (20%), autoimmune cytopenia (Coombs-positive hemolytic anemia), pneumonitis, nephritis, hepatitis, arthritis, myositis, alopecia are less common. However, cases of manifestation have been described over the age of one year, when patients did not have all the clinical and laboratory manifestations characteristic of severe forms of the disease. One of the main components of IPEX syndrome is polyendocrinopathy, manifested by the development of autoimmune diabetes mellitus, autoimmune thyroiditis. Immunological markers of endocrinopathies include antibodies to insulin (IAA), pancreatic islet cells (ICA), glutamate dehydrogenase (GAD), tyrosine phosphatase (IA-2), antibodies to the zinc transporter (ZNT8), antibodies to thyroid peroxidase and thyroglobulin. Other autoantibodies are also detected - to neutrophils, erythrocytes and platelets, antinuclear, antimitochondrial, antibodies to keratin, collagen, etc. In addition, for of this disease is characterized by the development of autoimmune enteropathy, clinically manifested by profuse watery diarrhea with the development of malabsorption syndrome, the immunological markers of which are antibodies to enterocytes (villin VAA and harmonin HAA). Increased IgE levels and an increase in the number of eosinophils are characteristic of patients with the classic severe form of the disease. Due to the severity of the disease and high mortality in this group of patients, it is extremely important early diagnosis and timely initiation of therapy. To date, the most effective treatment method is transplantation. bone marrow or allogeneic hematopoietic stem cell transplantation. In order to correct immunodeficiency, it is possible to use immunosuppressive monotherapy (cyclosporine A, tacrolimus) or combined therapy - a combination of immunosuppressive drugs with steroids. Sirolimus (rapamycin) has been shown to be effective, with several patients experiencing sustained remission of the disease. In addition to immunosuppressive therapy, replacement therapy endocrine disorders, adequate nutritional support, symptomatic therapy. Clinical case Patient K., born on April 19, 2016, at term with a weight of 2840 g and a body length of 51 cm. Delivery was performed by emergency cesarean section due to increasing intrauterine fetal hypoxia. Apgar score 7/7 points. From the anamnesis it is known that this is the fourth pregnancy, it occurred against the background of chronic genital infection, placental insufficiency, chronic gastritis, small weight gain, isthmic-cervical insufficiency, threat of interruption at 30 weeks. Previous pregnancies ended in spontaneous miscarriage early stages. The causes of miscarriage are unknown, the woman has not been examined. From birth, the child’s condition was severe due to respiratory disorders and signs of central nervous system (CNS) depression. Mechanical ventilation was performed; extubated when spontaneous breathing was restored. From the first day of life, an increase in blood sugar was detected to 10.4 mmol/l with an increase in dynamics to 29.0 mmol/l; according to CBS data, signs of metabolic acidosis were noted. An increase in glycemia levels was accompanied by glucosuria (sugar in the urine up to 2000 mg/dL) and ketonuria. A biochemical blood test showed signs of hyperenzymemia (ALT 87.8 U/L, AST 150 U/L). On the 2nd day of life, due to persistent hyperglycemia (maximum blood glucose level 33.6 mmol/l), intravenous administration of simple insulin was started at a rate of 0.03-0.1 U/kg/h, depending on blood glucose levels. Enteral nutrition with adapted mixtures was received in fractions through a tube. When daily monitoring of blood glucose levels during insulin therapy, significant variability of glycemia during the day was recorded from 1.7 to 22.0 mmol/l. On the 8th day of life, a deterioration in the condition was noted (signs of central nervous system depression, increasing respiratory failure, metabolic disorders associated with decompensation of carbohydrate metabolism, persistent low-grade fever up to 37.9 °C, bloating, repeated vomiting, diarrhea, trophic skin disorders in form of dryness and large-plate peeling). These symptoms were regarded as a manifestation of necrotizing enterocolitis (NEC 2a). For further examination and treatment, the patient was transferred to the Russian Research Center of the State Budgetary Healthcare Institution of the Children's Hospital of Petrozavodsk. During an examination at the Russian Research Center of the State Budgetary Healthcare Institution of the Children's Hospital, a series of clinical blood tests revealed a drop in hemoglobin level from 150 to 110 g/l, leukocytosis from 8.9 to 22.4 thousand, a change in the leukocyte formula due to an increase in the number of eosinophils from 5 to 31%, monocytes and blood platelets. According to the results of a biochemical blood test, hypoproteinemia was registered up to 36.4 g/l (N 49-69), a tendency towards hyponatremia 135 mmol/l (N 135-155) with normal indicators potassium 4.7 mmol/l (N 4.5-6.5), hyperenzymemia AlAT-87 U/l (N0-40) and AST 150 U/l (N0-40), increased CRP titer to 24.7 mg /m. There was no growth of microflora in the blood culture; enterococcus was found in the urine culture. Due to persistent hyperglycemia, the level of C-peptide was determined, which turned out to be reduced (0.1 nmol/l; N0.1-1.22 nmol/l). At ultrasound examination pneumatosis of the intestinal walls and an increase in the size of the pancreas (head - 9.0 mm, body - 9.0 mm, tail - 10.0 mm) were detected; a progression of sizes was observed over time. He received parenteral nutrition, infusion therapy aimed at eliminating electrolyte disturbances, antibacterial therapy, and insulin was administered intravenously. During treatment, electrolyte disturbances were eliminated, but it was not possible to achieve stabilization of glucose levels, and severe dyspeptic disorders also persisted. When attempting to restore enteral nutrition, abdominal bloating, vomiting and diarrhea appeared. On the 19th day of life, the boy was taken to the intensive care unit in extremely serious condition. perinatal center Clinics of St. Petersburg State Pediatric Medical University. Upon admission, the child had a sluggish, unemotional cry, spontaneous motor activity and muscle tone were reduced, the newborn's reflexes were weak. Fever (body temperature 38.1 °C). Large fontanel 1.0 × 1.0 cm, sunken. The skin is pale, dry, reduced turgor, large-plate peeling. On auscultation, breathing was harsh and carried out evenly in all parts of the lungs; respiratory rate 46 per minute. Heart sounds were rhythmic, slightly muffled; heart rate - 156 per minute. Noticeable bloating of the abdomen and moderate enlargement of the liver. The rate of diuresis is 7-8 ml/kg/h (against the background of ongoing infusion therapy). Watery stools 7-9 times a day. The dynamics of weight gain was negative (birth weight - 2840 g, upon admission to the clinic of St. Petersburg State Pediatric Medical University - 2668 g). In the intensive care unit, infusion therapy was continued, aimed at eliminating electrolyte disturbances. Hyponatremia was difficult to correct due to compensatory mechanism balance of blood osmolarity in response to hyperglycemia, accompanied by polyuria up to 7-8 ml/kg/h and loss of sodium in the urine, as well as severe enteropathy (stool 6-10 times a day, copious, liquid up to 350 ml/day). Insulin was received intravenously at 0.01-0.04 U/kg/h depending on blood glucose levels. Partial enteral nutrition was carried out with a pasteurized hydrolyzed mixture with a gradual transition from tube feeding to independent fractional feeding against the background of stabilization of the condition. On the 28th day of life, he was transferred to the department of pathology of newborns and infants, where examination and treatment were continued. During the examination, anemia was noted in the hemogram (hemoglobin - 93 g/l, erythrocytes - 2.91 ∙ 1012/l); leukocytosis (up to 28.7 ∙ 109/l), severe eosinophilia (up to 61%); in the biochemical blood test there was hypoproteinemia (total protein - 38.4 g/l). When studying blood hormone levels, it was re-identified low level C-peptide - 0.5 ng/ml (N 0.1-1.22 nmol/l); thyroid hormones were normal (free T4 - 14.8 pmol/l (N 10.0-23.2); TSH - 6.28 µU/ml (N 0.23-10.0). The combination of neonatal diabetes diabetes, enteropathy, specific skin manifestations and signs of chronic recurrent infection (febrile fever, increase in leukocytosis upon withdrawal antibacterial therapy) made it possible to suspect IPEX syndrome in the patient, the structure of which includes immunodeficiency. To clarify the nature of polyendocrinopathy, a study was conducted aimed at searching for immunological markers. As a result, a high titer of antibodies to thyroid peroxidase (243.9 IU/ml; N 0-30) and antibodies to the islets of Langerhans in a positive titer (antibodies to GAD1.29 U/ml; N 0-1.0), a titer of antibodies to insulin was 5.5 U/ml (N 0.0-10.0). Antibodies to steroid-producing adrenal cells were not detected. Thus, the autoimmune nature of diabetes mellitus was proven and autoimmune thyroiditis was diagnosed. Taking into account the presence of clinical and laboratory signs of immunodeficiency, an in-depth immunological examination was performed and a high level of IgE was detected (573.6 IU/ml, the norm is 0-15). A molecular genetic study (multigene targeted sequencing) was carried out, which revealed a mutation in the FOXP3 gene, confirming the presence of IPEX syndrome in the child. At the age of two months, four days, due to the high risk of sepsis and the threat fatal outcome the boy was transferred to the Federal State Budgetary Institution "FNKTs DGOI named after. Dmitry Rogachev" of the Russian Ministry of Health for immunosuppressive therapy and bone marrow transplant surgery. In the presented clinical case, a previously undescribed mutation of the FOXP3 gene c.1190G > T (p.Arg397Leu) was identified in a child with neonatal diabetes mellitus, primary immunodeficiency and severe enteropathy. DNA analysis of the patient's mother revealed the same damage in a heterozygous state. The detected variant is localized in the DNA-binding C-terminal forkhead domain and is considered pathogenic by the main predictive programs (Polyphen2, SIFT, Mutation Taster). A significant indirect argument in favor of the pathogenicity of the identified variant is the fact that mutations c. 1189C > T and s. 1190G>A, affecting the same codon 397, were previously found in patients with IPEX syndrome. It is known that mothers of patients with IPEX syndrome are characterized by multiple episodes of spontaneous abortions when carrying male fetuses. In the described case, the medical history of the patient's mother (carrier of the mutation) was also aggravated by early pregnancy loss. This further confirms that the new mutation we discovered has a serious effect on the function of FOXP3, causing increased embryonic lethality. Genetic verification of neonatal diabetes mellitus is extremely useful, as it allows one to clarify the nature of the disease and choose the optimal treatment tactics. This is especially true for patients with syndromic forms, which usually have severe course. Detection of a mutation in a sick child is very important for medical genetic counseling of the family, since it makes it possible to conduct prenatal DNA diagnostics in the case of subsequent pregnancies. Given clinical case indicates that the interaction of doctors of various specialties (neonatologists, endocrinologists, gastroenterologists, immunologists, geneticists) contributes to more effective establishment of the correct diagnosis in patients with rare diseases.
Maria E Turkunova
St Petersburg State Pediatric Medical University, Ministry of Healthcare of the Russian FederationAutoantibodies to Harmonin and Villin Are Diagnostic Markers in Children with IPEX Syndrome
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826762/
Conceived and designed experiments: V.L. Bosi R.B. Performed the experiments: CL E. Bazzigaluppi CB. Analyzed data: VL LP FB RB E. Bosi. Reagents/materials/analysis tools used: LP FB. Wrote the article: E. Bosi. Contributed to writing/editing the manuscript: VL LP FB RB.
Autoantibodies to conjunctival enterocyte antigens (75 kDa USH1C protein) and villous (95 kDa actin binding protein) are associated with the syndrome of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). In this study, we assessed the diagnostic value of harmonic and villous autoantibodies in IPEX and IPEX-like syndromes. Harmonin and villin autoantibodies were measured by a novel quantitative luminescence immunoprecipitation system (LIPS) assay in patients with IPEX, IPEX-like syndrome, primary immunodeficiencies (PID) with enteropathy, all diagnosed by FOXP3 gene sequencing, and type 1 diabetes (T1D), celiac disease and healthy blood donors as control groups. Harmonin and villin autoantibodies were detected in 12 (92%) and 6 (46%) of 13 IPEX patients and in none of the IPEX, PID, T1D, and celiac disease patients, respectively. All IPEX patients, including one case with a late and atypical clinical presentation, had either harmonic and/or villous autoantibodies or tested positive for anti-enterocyte antibodies by indirect immunofluorescence. When measured in IPEX patients in remission after immunosuppressive therapy or hematopoietic stem cell transplantation, harmonic and villous autoantibodies became undetectable or persisted at low titers in all cases, but in one in which harmonic autoantibodies remained persistently high. In one patient, the peak of harmonic antibodies paralleled the relapse phase of enteropathy. Our study demonstrates that harmonic and villous autoantibodies measured by LIPS are sensitive and specific markers of IPEX, differentiate IPEX, including atypical cases, from other early childhood enteropathy-related disorders, and are useful for screening and clinical monitoring of affected children.
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is a monogenic autoimmune disease characterized by severe enteropathy, type 1 diabetes (T1D) and eczema. The syndrome is caused by mutations in the FOXP3 gene, which is responsible for serious violation regulatory T (Treg) cells. While genetic analysis is the method of choice for definitive diagnosis, there is no clear genotype-phenotype correlation, and the course of the disease varies among patients. Additionally, despite the classification of IPEX as an immunodeficiency disorder, there are no clear immunological parameters that predict disease severity or response to therapy. Additionally, disorders with a similar clinical phenotype, called IPEX-like syndromes, can exist in the absence of FOXP3 mutations, presenting challenges for clinical management and choice of therapeutic agents - . Therefore, identifying markers specifically associated with IPEX immune dysfunction would be extremely useful for diagnostic purposes. Circulating enterocyte autoantibodies detected by indirect immunofluorescence have been described in the past in association with various enteropathies, including those eventually identified as IFEX syndrome, but molecular targets These serological markers have long been unknown. A distinct enterocyte autoantigen recognized by IPEX patient sera was then identified as the 75 kDa AIE-75 protein, and further characterized as Usher Syncrome protein (USH1C), also known as harmonic, a protein that is reported to be part of supramolecular protein networks, connecting transmembrane proteins to the cytoskeleton in photoreceptor cells and hair cells inner ear. Harmonine autoantibodies (HAA), detected by immunoblotting and radioligand, have been reported in patients with IPEX and in a small proportion of patients with colon cancer. More recently, an actin-binding protein designated actin 95 kDa, involved in the organization of the actin cytoskeleton at the brush border of epithelial cells, has been described as an additional target of autoantibodies in a subset of patients with IPEX. In contrast, to our knowledge, no information has been reported on either HAAs or villous autoantibodies (VAAs) in IPEX-like syndromes, primary immunodeficiencies (PID) with enteropathy, or in disorders often associated with IPEX, such as T1D and the autoimmune enteropathies of various types. origin.
The aim of this study was to develop quantitative assays to measure HAA and VAA based on the newly developed Luminescent Immuno Precipitation System (LIPS), determine their diagnostic accuracy in IPEX, IPEX-like and PID syndromes, evaluate them according to enterocyte antibodies tested by immunofluorescence, and evaluate their value in the clinical follow-up of IPEX patients.
Thirteen patients with IPEX and 14 patients with IPEX-like syndrome were tested at LIPS for the presence of HAAs and VAAs. As controls, we studied 5 patients with PID of various origins [two with CD25 deficiency, two with Wiskott Aldrich syndrome (WAS)] and one with adenosine deaminase deficiency-severe combined immunodeficiency disorder (ADA-SCID), all conditions characterized by early onset enteropathy] , 123 with T1D, 70 with celiac disease and 123 healthy blood donors. The diagnosis of IPEX was based on clinical and molecular findings according to the criteria established by the Italian Association of Pediatric Hematology and Oncology (AIEOP, www.AIEOP.org). Mutations and clinical data of IPEX and IPEX patients are summarized in Tables S1 and S2, respectively. All IPEX patients except Pt19, Pt21, Pt22 and Pt24 have been described in previous publications. PT24 is an atypical form of the disease characterized by late onset, without signs of enteropathy, but severe gastritis in the presence of inflammatory mucosal infiltrates associated with villous atrophy. General level IgG was available in 10 of the 13 IPEX patients studied: of these, 8 were in normal range by age (with only one patient on intravenous (IV) Ig therapy), whereas in two cases they were slightly increased. Patients diagnosed with IPEX-like syndrome had clinical manifestations of IPEX but tested negative for mutations in the FOXP3 gene. Patients like IPEX presented at least one of the main clinical features IPEX (autoimmune enteropathy and/or T1D) associated with one or more of the following autoimmune or immune-mediated diseases: dermatitis, thyroiditis, hemolytic anemia, thrombocytopenia, nephropathy, hepatitis, alopecia, hyper IgE with or without eosinophilia. Clinical and laboratory parameters excluded other monogenic diseases such as WAS, Omenn syndrome, hyper IgE syndrome, and autoimmune lymphoproliferative syndrome. At least one serum sample from patients with IPEX and IPEX-like syndromes was available for autoantibody testing at the time of diagnosis. In six IPEX patients, multiple serum samples were also obtained during clinical follow-up and used for additional autoantibody measurements to examine correlation with clinical outcome (Pt12:8 samples from birth to 8 years, Pt14:7 samples from 6 months to 13 years, samples Pt17-3, from 4 months to 3.5 years, samples Pt19:44 from 4 months to 2 years, samples Pt22:3 from 0 to 5 months; Pt 23:44 from 4 to 10 years). All patients with PID were diagnosed based on molecular testing. Patients with T1D were all recent cases, with a diagnosis based on American Diabetes Association criteria; patients with celiac disease were studied at the time of diagnosis based on jejunal biochemistry.
According to the Helsinki statement, written informed consent was provided to each patient and next of kin, guardian or guardian on behalf of the minors/children participating in this study. The study was approved by the local research ethics committee in San Raffaele.
All patients classified as having IPEX or IPEX-like syndrome were entered for FOXP3 mutations. Genomic DNA was isolated from peripheral blood using the phenol-chloroform method or the QIAamp DNA Blood Mini Kit (Qiagen) following the manufacturer's instructions. Eleven exons, including all intron-exon boundaries, were amplified from genomic DNA by PCR with specific flanking intronic primer pairs. Amplified gene fragments were sequenced using the BigDye Terminator cycling kit (Applied Biosystems) on an ABI PRISM 3130xl automated genetic analyzer and an ABIPRISM 3730 genetic analyzer (Applied Biosystems).
The Renilla luciferase coding sequence was cloned into the pTnT plasmid (Promega, Milan, Italy) to generate the pTnT-Rluc vector. Full-length harmonic and villus DNA coding sequences were then amplified by RT-PCR and cloned separately into pTnT-Rluc downstream and in frame with Renilla lumiferase. Recombinant chimeric Rluc-Harmonin and Rluc-Villin were expressed by in vitro -coupled transcription and translation using the pTnT-quick SPM rabbit reticulocyte lysate free cell system (Promega). To test for the presence of HAAs or VAAs, Rluc-Harmonin and Rluc-Villin were used as antigens in LIPS (17) by incubating 4 × 106 light units equivalents with 1 μl of each patient's serum in PBS pH 7.4-Tween 0.1% (PBST) for 2 hours at room temperature, IgG immune complexes were isolated by adding protein A-Sepharose (GE Healthcare, Milan, Italy) followed by incubation for 1 hour at 4°C and washing with PBST of unbound Ag by filtration in 96-well Costar filter plates 3504 (Corning Life Sciences, Tewksbury, USA). Immunoprecipitated antigens were then quantified by measuring the recovered luciferase activity after adding Renilla lumiferase substrate (Promega) and measuring light emission for 2 seconds in a Centro XS3 luminometer (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Results were expressed in arbitrary units derived from either the antibody index (BAA) using positive and negative sera according to the formula (cps control serum-cps-negative serum) / (cps-positive serum-cps-negative serum) x100, or from a standard curve (HAA), consisting of serial dilutions of positive starting serum. The positivity cutoff was set at the 99th percentile of values observed in healthy blood donors, as is common practice in T1D-sensitive autoantibody workshops analyzing sensitivity and specificity.
Enterocyte autoantibodies were determined in IPEX and IPEX-like patient groups by indirect immunofluorescence on cryostat sections of normal human or monkey jejunum as previously described.
Markers of T1D and celiac disease autoantibodies, including glutamic acid decarboxylase (GADA) antibodies, insulinoma-associated protein 2, insulin, zinc transporter 8, and transglutaminase-C, were measured in all IPEX, IPEX-like, PID, T1D, celiac, and healthy controls donor control groups by immunoprecipitation using LIPS or radiolens as previously described. All results were expressed in arbitrary units derived from standard curves obtained by serial dilution of positive stock sera.
This study used only descriptive statistics. Calculation of the 99th percentile of random units in blood donors for threshold selection was performed using Stata (StataCorp LP, USA). Conditional probability of testing positive (sensitivity) or negative (specificity) for a HAA or VAA depending on the presence or absence of the IPEX disease state and associated confidence intervals 95% was calculated using the Vassar Stats website for statistical computing (http://vassarstats.net/clin1.html). The correlation between HAA and VAA titers was based on Spearman's rank correlation tests and was calculated using Graphpad Prism 5 software.
Elevated circulating HAA concentrations were found in 12 of 13 (92%) patients with IPEX, whereas they were negative in patients with IPEX, PID, T1D, and celiac disease (Figure 1A). Elevated concentrations of circulating VAAs were found in 6 (46%) IPEX patients (Pt19, Pt14, Pt12, Pt17, Pt3, Pt21, with the last four having titers equal to or greater than 98 VAA AU), including a patient without HAAs (Pt17), then how VAAs were negative in IPEX-like and other disease control groups (Fig. 1B). All patients with IPEX were positive for either HAA or VAA, giving the combination of HAA and VAA a test sensitivity of 100% (95% CI: 71.6 to 100%) and test specificity of 97.6% (95% CI: 92.5 to 99.4%) for diagnosing IPEX syndrome. No clinical or phenotypic characteristics correlated with the presence of either autoantibodies in patients with IPEX. No significant correlation was observed in IPEX patients between titanium autoantibodies to AAA and VAA (Spearman r = -0.3 p = ns). GADA, as the most common T1D autoantibodies, were found in patients with IPEX (9 of 13, 5 with T1D), IPEX type syndrome (4 of 14, 2 with T1D), and PID (3 of 5, 1 with T1D) (Fig. 1C). Other T1D autoantibodies were detected in lower proportions, including insulin autoantibodies in 5 IPEX, 4 IPEX-like, and 2 PID, and Zinc Transporter 8 autoantibodies in one IPEX patient. There was no correlation between GADA and HAA or VAA titers (Spearman r = -0.017 p = ns and r = 0.34 p = ns, respectively). None of the patients with IPEX, IPEX-like syndrome, or PID had celiac disease-associated tissue transglutaminase-C IgA or IgG autoantibodies (data not shown).
HAA (panel A), VAA (panel B) and GADA (panel C) serum IgG titers expressed in arbitrary units in IPEX (n = 13), IPEX-like (n = 14), PID (n = 5), T1D (VAA and GADA n = 123, VAA n = 46), patients with celiac disease (HAA n = 70, VAA n = 46, GADA n = 44) and controls (HAA and VAA n = 123, GADA n = 67). The dotted line indicates the cutoff for positivity.
All IPEX sera, but one (Pt 22), 10 IPEX-like and 3 PID sera were tested for enterocyte antibodies by immunofluorescence on intestinal cryostat sections. All IPEX patients tested were positive for enterocyte antibodies. HAA positive sera showed strong reactivity against intestinal enterocytes and the cytosol with the greatest intensity at the border of the brush (Fig. 2A). Isolated high titer VAA showed strong staining to clear the border but not the cytosol (Fig. 2B). Outside the IPEX patient group, only one serum from a patient with PID infection with a CD25 gene mutation and negative for HAA and VAA (Pt L1) showed positive staining of enterocytes limited to the border of the hand (Fig. 2C).
HAA from IPEX Pt 19 binds the brush border and enterocyte cytosol (panel A), while VAA from IPEX Pt 17 binds only the brush border (panel B). IgG from PID Pt L1 binds the enterocyte brush border (panel C). Lack of binding in IPEX-like Pt L30 (panel D).
Consecutive samples for HAA and VAA measurements were available for 6 IPEX patients (Pt12, Pt14, Pt17, Pt19, Pt22 and Pt23): all of whom had undergone hematopoietic stem cell transplantation (HSCT) as therapeutic therapy preceded in 4 cases by a variable period of systemic immunosuppression. At the time of this report (April 2013), all but two transplanted patients were alive, in clinical remission from their enteropathy, and not on immunosuppressive therapy (Table S1). Genetic analysis of peripheral blood collected after transplantation showed 100% donor chimerism in 4 cases (Pt12, Pt14, Pt19 and Pt22) and mixed donor/recipient chimerism in other patients. At the onset of enteropathy, three patients had both HAA and VAA (Pt12, Pt14, and Pt19), one had only VAA (Pt17), and two had only HAA (Pt22 and Pt23) (Fig. 3). In five cases (Pt12, Pt14, Pt17, Pt22 and Pt23), clinical remission or marked improvement after either immunosuppression or HSCT was accompanied by a decrease in both HAA and/or VAA titers, which became undetectable or persisted at very low titers in four cases with longest observation. In one case (Pt19), after HSCT VAA became undetectable, while HAA persisted at high titers despite clinical remission (Figure 3D). In at least one case (Pt14), HAA was found to be a sensitive marker of enteropathy: HAA was detected at high titers in association with severe enteropathy at the time of IPEX diagnosis, and then decreased during clinical and histological remission after immunosuppressive therapy, peaking during clinical relapse , and then became persistently undetectable after successful HSCT and clinical remission (Figure 3B). Although less prevalent, VAA showed a pattern similar to that of HAA. The fall in autoantibodies observed after HSCT was caused by B cell and IgG deficiency secondary to conditioning. Indeed, with the exception of Pt22, who had a short postoperative transplant, all patients with reduced HAA or VAA titers after HSCT (Pt12, 17, and 23) were already immunized and IVIg therapy was independent at the time of the first post-HSCT.
The vertical axis denotes HAA (diamonds) and VAA (triangles), autoantibody titers expressed in arbitrary units, along the horizontal axis in months. The vertical dotted line indicates the HSCT date, the horizontal dotted and dotted lines indicate the cutoff for HAA and VAA positivity, respectively.
In this study, we show that HAA and VAA, easily measured by new LIPS assays and used in combination, are highly sensitive and specific markers of IPEX syndrome and can predict its clinical outcome. In fact, all IPEX patients with a diagnosis confirmed by genetic testing had elevated concentrations of HAAs or VAAs. In contrast, none of the patients with enteropathy without FOXP3 mutations (eg, IPEX-like or PID), patients with T1D or celiac disease were positive for either HAA or VAA. Of the two markers, HAA had the highest sensitivity, found in 12 of 13 IPEX patients, whereas VAA was detected in only six of them. Notably, HAA and VAA have proven to be valuable markers of IPEX syndrome also in atypical cases such as Pt24, where enteropathy was not part of the clinical presentation, instead severe gastritis was dominant, in which IPEX was suspected and then confirmed by FOXP3 gene sequencing only after detection of elevated HAA . In the future, the new LIPS assay will allow for more systematic screening of HAA and VAA in patients with heterogeneous clinical syndromes, with the ability to identify more cases of clinically atypical IPEX syndromes.
GADAs were the second most common autoantibody reaction observed in IPEX patients after HAAs. Although GADAs are the most common marker of T1D autoantibodies, with wide range titer during clinical onset, they are not always associated with diabetes. In fact, they can be found in other autoimmune diseases, including Stiff Person Syndrome and Autoimmune Polyendocrinopathy (APS). Interestingly, in patients with AFP, GADA is more correlated with the development of gastrointestinal symptoms rather than with diabetes. Intriguingly, also in our IPEX patients, GADAs were largely common without being consistently associated with T1D.
In addition to being accurate markers of IPEX syndrome, HAA and VAA may have potential prognostic value, especially with regard to associated enteropathy. Six patients with available post-challenge samples had high titers of both HAA and VAA at the time of diagnosis or at the onset of gastrointestinal symptoms and before treatment. Thereafter, in five cases after immunosuppressive treatment and/or HSCT (Pt12, Pt14, Pt17, Pt22, and Pt23), HAA and VAA titers decreased to become undetectable or remained at low titers around the detection threshold, reflecting clinical and histological remission of the associated enteropathy. In one of them (Pt14), a transient relapse of enteropathy occurring during immunosuppressive treatment was accompanied by a peak in HAA followed by a decline after clinical remission. Unfortunately, in this patient, the lack of serial samples prevented us from documenting the timing of autoantibody increases preceding relapse of enteropathy. In one case (Pt19), clinical remission was accompanied by a decrease in VAA, but not HAA, which persisted at high titers up to 15 months after HSCT. The finding of decreased HAA and VAA titers after HSCT in most but not all patients is extremely intriguing, possibly related to the survival of residual host lymphocytes or plasmacells responsible for the persistent production of these autoantibodies.
Introduction of these autoantibody markers into clinical practice would be relatively simple given the ease of their measurement using the recently developed LIPS. Recently, this technology has been proposed as a new non-radioactive procedure to replace radioactive antibody and immunoprecipitate protein-A into gold standard in vitro transcribed and translated 35S-methionine-labeled recombinant human antigens, validated through established autoantibody standardization programs in both T1D and celiac disease. In recent reports, LIPS has demonstrated performance comparable to radiolink assays and higher than the previously existing ELISA. In this study, LIPS was developed using recombinant chimeric Renilla lumiferase (Rluc)-Harmonin and Rluc-Villin as antigens, resulting in an assay with low background noise and linear quantitative autoantibody measurements capable of distinguishing positive results from negative serum samples. In summary, measurement of HAA and VAA using LIPS has proven to be a rapid, simple, and reproducible test that is easily applicable for clinical use.
Interestingly, the same diagnostic performance of combined HAAs and VAAs was observed with enterocyte autoantibodies detected by conventional indirect immunofluorescence. It also remains unclear, but worth testing in the future, whether harmonics and villi are the only antigens recognized on enterocytes by IPEX-associated autoantibodies, or if other enterocyte autoantigen targets of IPEX-associated antibodies have not yet been identified.
Until now, IPEX was considered a T cell, namely immune disease Treg cell dysfunction, with limited attention to associated defects in the humoral immune response: Our results highlight the association of major FOXP3 gene mutations with a robust and quantifiable antigen-specific autoantibody. However, since B cells do not express FOXP3, FOXP3 mutations are unlikely to have a direct impact on B cell development and/or antibody production. However, recent studies indicate that B cells may be both direct and indirect targets of Treg cell-mediated suppressive function, and alteration of Treg cells influences autoantibody titers in both mouse models and humans - Moreover, Direct evidence from foxp3 mutant mice indicates that the absence of Treg cells is associated with abnormal B cell development, loss of allergic B cells, and development of long-lived plasma cells. Additionally, it has recently been demonstrated that in humans, FOXP3 deficiency results in the accumulation of autoreactive clones in the mature naïve B cell compartment, suggesting an important role for Treg cells in checkpoint control from a peripheral B cell perspective.
The mechanisms responsible for hormone and villous autoimmunization in IPEX and the role of these autoantigens in the pathological manifestations of IPEX syndrome remain unknown. Harmonin is expressed in several tissues, including small intestine, colon, kidney, eye, inner ear vestibule and, weakly, pancreas. In the intestine, harmonizer expression is predominantly found in luminal surface epithelial cells and in the upper half of the intestinal crypts—and is likely localized to hand microvilli; similar localization has been shown for villi,. Given that the main histopathological feature of IPEX enteropathy is villous atrophy with apoptotic cell death of intestinal epithelial cells combined with moderate to severe inflammation, it is likely that in this context, harmonica and villous tissue may act as relevant molecular targets of pathogenic autoimmunity.
This study showed that HAA and VAA measured by LIPS are accurate diagnostic markers of IPEX syndrome, with 100% concordance of FOXP3 gene mutations that differentiate IPEX, including atypical cases, from other childhood enteropathy-related disorders. Overall, these data indicate that HAA and VAA should be included in the diagnostic flow and clinical observation monitoring patients with IPEX syndrome in whom changes in HAA and VAA titers suggestive of recurrent enteropathy may help clinicians make rapid therapeutic decisions.
Clinical features of IPEX patients.
Clinical features of IPEX-like patients.
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The authors are grateful to their colleagues who kindly provided serum samples and clinical information about their IPEX and IPEX patients: E. S. Kang and Y. H. Choe, Seoul, Republic of Korea; G. Zuin, Milan, Italy; A. Staiano, R. Troncone and V. Discepolo, Naples, Italy; J. Schmidtko, Berne, Switzerland; A. IkinciogullariZ. SedaUyan, M. Aydogan, E. O. Tzu, Ankara, Türkiye; G.R. Corazza and R. Ciccocioppo, Pavia, Italy; S. Vignola, Genoa, Italy; A. Bilbao and S. Sanchez-Ramon, Madrid, Spain; J. Reichenbachand M. Hoernes, Zurich, Switzerland; M. Abinun and M. Slatter, Newcastle upon Tyne, U.K.; M. Cipolli, Verona, Italy; F. Gurakan, Ankara, Türkiye; F. Locatelli and B. Lucarelli, Rome, Italy; C. Cancrini and S. Corrente, Rome, Italy; A. Tommasini, Trieste, Italy; L. Guidi, Rome, Italy; E. Richmond Padilla and O. Porras, San Jose, Costa Rica; S. Martino and D. Montin, Turin, Italy; M. Hauschild, Germany; K. Nadeau and M. Butte, Stanford, CA; A. Aiuti, G. Barera, F. Meschi and R. Bonfanti, Milan, Italy. The authors also thank: M. Cecconiand D. Coviello for FOXP3 genotyping; and members of the Italian IPEX research group (www.ipexconsortium.org) R. Badolato, M. Cecconi, G. Colarusso, D. Coviello, E. Gambinera and A. Tommasini for support and encouragement. The authors thank the patients and their families for their trust and participation in our studies.
IPEX Syndrome is a syndrome of immune dysregulation, polyendocrinopathy, and enteropathy with an X-linked recessive type of inheritance (Immunodysregiilation, Polyendocrinopathy, and Enteropathy, X-Linked).
Symptoms: Polyendocrinopathy (a disorder in the endocrine gland system), manifested by the development of type 1 diabetes mellitus. In this type of diabetes, immune cells attack and destroy pancreatic cells that produce insulin, a hormone involved in the metabolism of glucose (sugar) in the body. In patients with IPEX, insulin is not produced and a state of hyperglycemia develops - high content blood sugar. It is also possible to develop autoimmune thyroiditis - inflammation of the thyroid gland caused by an attack on one's own immune system; the thyroid gland can no longer perform its functions properly (for example, calcium metabolism in the body is disrupted). Enteropathy (damage to the gastrointestinal tract) is manifested by persistent diarrhea that begins before or during consumption of food, possible intestinal bleeding. Hemolytic anemia - hemolysis (destruction) of red blood cells and a decrease in the amount of hemoglobin. Skin rashes according to the type of eczema (skin rash accompanied by itching and peeling). Arthritis (inflammation of the joints), lymphadenopathy (enlargement and pain lymph nodes), kidney damage. Cachexia ( extreme degree exhaustion). Increased susceptibility to infections due to the presence of immune dysregulation (impaired interaction of immune system cells with each other and with other cells) and/or neutropenia (reduction in the number of neutrophils - cells of the immune system, the main function of which is protection against infections): pneumonia (inflammation lungs), peritonitis ( purulent inflammation peritoneum), sepsis (blood poisoning), septic arthritis(purulent inflammation of the joints).
IPEX syndrome is associated with mutations in the FOXP3 gene
Research method: FOXP3 gene sequencing
syndrome protein), which plays an important role in the functioning of the cytoskeleton. It regulates actin polymerization. The normal function of this protein is necessary for full cell motility, their polarization, filopodia formation during chemotaxis, cell adhesion and the formation of immune synapse during the interaction of cells of the immune system.
Depending on the location of the mutations and the extent of the gene region affected by them, three clinical variants of the disease develop: full-blown Wiskott-Aldrich syndrome (a consequence of deletions) and variants with isolated manifestations of thrombocytopenia or neutropenia. The classic presentation of Wiskott–Aldrich syndrome is characterized by thrombocytopenia with small platelets, eczema, and recurrent infections.
Wiskott–Aldrich syndrome is characterized by multiple disorders in the immune system, affecting predominantly the phagocytic and cytolytic activity of innate immune cells, i.e. functions that are most dependent on cell movement and the active participation of the cytoskeleton. Disruption of the formation of the immune synapse between T lymphocytes and APCs affects all manifestations of adaptive immunity.
Ataxia-telangiectasia (Louis-Bar syndrome)
A hereditary disease caused by a defect in the ATM gene (Ataxia telangiectasia mutated). Refers to diseases based on the syndrome of chromosomal breakdowns. The disease develops as a result of mutations that occur in any part of the ATM gene. The result of mutations can be a complete absence or weakening of ATM protein synthesis, as well as the synthesis of a functionally defective protein.
The ATM protein is a serine threonine protein kinase. Its main function is to initiate signals for the repair of double-stranded DNA breaks that occur both under physiological conditions (during meiosis, rearrangement of V genes of antigen recognition receptors, etc.) and induced by the action of external factors(for example, ionizing radiation). When DNA breaks occur, ATM kinase autophosphorylates and changes from dimeric to monomeric form. ATM kinase ensures phosphorylation of proteins of the MRN complex and associated factors that directly carry out DNA repair. In the case of a small number of ruptures, they successfully perform this function. If successful repair is impossible, apoptosis develops, triggered by the p53 factor. The lack of complete DNA repair causes instability of the genome, resulting in an increase in the radiosensitivity of cells and the frequency of development malignant tumors, especially lymphomas and leukemias.
The most characteristic clinical sign of ataxia-telangiectasia is increasing ataxia, manifested by changes in gait. It is caused by neurodegeneration with cerebellar atrophy. The development of neurodegenerative processes is associated with the fact that during the maturation of brain neurons, DNA recombination processes occur, accompanied by double breaks. Another symptom that gives the disease its name, telangiectasia, is a persistent dilatation of the ocular and facial blood vessels.
Impaired repair of DNA breaks that occur during the maturation of T and B lymphocytes also underlies the immunodeficiency observed in ataxia-telangiectasia. Immunodeficiency manifests itself in chronic recurrent bacterial and viral infectious diseases of the bronchopulmonary apparatus, which usually causes the death of the patient.
Nijmegen syndrome
Nijmegen is the city in Holland where the syndrome was first described. This hereditary disease is classified as a syndrome of chromosomal breakdowns accompanied by the formation of genome instability. The development of this disease is associated with a mutation in the NBS1 gene, the product of which, nibrin, is involved in DNA repair as part of the MRN complex, being a substrate for phosphorylation by the ATM protein kinase. In this regard, both the pathogenesis and clinical manifestations of Nijmegen syndrome practically coincide with those of ataxia-telangiectasia. In both cases, neurodegenerative changes develop, however, with Nijmegen syndrome, microcephaly phenomena predominate, since DNA recombination processes also occur during the maturation of brain neurons.
Autoimmune lymphoproliferative syndrome
The disease is characterized by impaired apoptosis and associated non-malignant lymphoproliferation, hyperimmunoglobulinemia, autoimmune processes, and an increase in the content of CD3+ CD4- CD8- cells in the blood. The mutations underlying the syndrome are most often localized in the TFRRSF6 gene, which encodes the Fas receptor (CD95). Only mutations that cause changes in the intracellular region of the CD95 molecule lead to clinical manifestations. Less commonly, mutations affect the Fas ligand and caspase 8 and 10 genes (see section 3.4.1.5). Mutations are manifested by weakened expression of molecules encoded by the corresponding gene and weakening or complete absence apoptotic signal transmission.
X-linked lymphoproliferative syndrome
A rare immunodeficiency characterized by a perverted antiviral, antitumor and immune response. The causative agent of X-linked lymphoproliferative syndrome is the Epstein–Barr virus. The virus enters B cells through the interaction of the gp150 molecule of the viral envelope with the CD21 receptor on the cell membrane. In patients with X-linked lymphoproliferative syndrome, polyclonal activation of B cells occurs and unimpeded viral replication occurs.
Infection with the Epstein–Barr virus in X-linked lymphoproliferative syndrome is the result of a mutation in the SH2D1A gene, encoding the adapter protein SAP [ Signaling lymphocytic activation molecule (SLAM)- associated protein]. The SH2 domain of the SAP protein recognizes a tyrosine motif in the cytoplasmic part of SLAM and a number of other molecules. Processes that develop in cells of the immune system upon activation mediated through the SLAM receptor play a leading role in antiviral immunity. The SLAM receptor is expressed on thymocytes, T-, B-dendritic cells, and macrophages. Expression increases when cells are activated. The regulatory effect of the SAP protein is associated with the suppression of the activity of tyrosine phosphatases in
4.7. Immunodeficiencies | |
regarding SLAM. In the absence of SAP, SH-2 phosphatase binds freely to the SLAM receptor, dephosphorylates it, and inhibits signal transduction. The main effectors of antiviral defense, T and NK cells, are not activated, which leads to uncontrolled proliferation of the Epstein–Barr virus. In addition, SAP facilitates the interaction of the Fyn tyrosine kinase with the SLAM receptor, which facilitates the transmission of the activation signal.
In the diverse clinical manifestations of X-linked lymphoproliferative syndrome, the most consistent is fulminant infectious mononucleosis, benign and malignant lymphoproliferative disorders, as well as dysgammaglobulinemia or hypogammaglobulinemia. Among local lesions, liver damage predominates, caused by infiltration of Epstein-Barr virus-infected B cells and activated T cells, which leads to necrosis of liver tissue. Liver failure is one of the main causes of death in patients with X-linked lymphoproliferative syndrome.
IPEX syndrome
X-linked syndrome of immune dysregulation, polyendocrinopathy and enteropathy ( Immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome) develops as a consequence of mutations in the FOXP3 gene, localized on the X chromosome. FOXP3 is a “master gene” responsible for the development of regulatory T cells of the CD4+ CD25+ phenotype. These cells play a central role in restraining the activity of autospecific T-lymphocyte clones in the periphery. A defect in the FOXP3 gene is associated with the absence or deficiency of these cells and the disinhibition of various autoimmune and allergic processes.
IPEX syndrome manifests itself in the development of multiple autoimmune lesions of endocrine organs, digestive tract and reproductive system. This disease begins at an early age and is characterized by damage to a number of endocrine organs (diabetes mellitus type I, thyroiditis) with high level autoantibodies, severe enteropathy, cachexia, short stature, allergic manifestations(eczema, food allergies, eosinophilia, increased IgE levels), as well as hematological changes (hemolytic anemia, thrombocytopenia). Sick children (boys) die during the first year of life from repeated severe infectious diseases.
APECED syndrome
Autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy ( Autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy) is an autoimmune syndrome caused by a defect in negative selection of thymocytes. Its cause is mutations of the AIRE gene, responsible for the ectopic expression of organ-specific proteins in the epithelial and dendritic cells of the medulla of the thymus, responsible for negative selection (see section 3.2.3.4). The autoimmune process primarily affects the parathyroid glands and adrenal glands, as well as the islets of the pancreas (type I diabetes develops), the thyroid gland, and the genitals.
Often accompanied by the development of candidiasis. Defects in the morphogenesis of ectoderm derivatives are also revealed.
When considering the spectrum of primary immunodeficiencies, attention is drawn to the absence of nosological units associated with the pathology of NK cells. To date, just over a dozen mutations affecting the function of these cells in individuals have been described, suggesting that immunodeficiencies selectively affecting NK cells are extremely rare.
4.7.2. HIV infection and acquired immunodeficiency syndrome
In addition to primary immunodeficiencies, the only disease for which damage to the immune system is the basis of pathogenesis and determines symptoms is acquired immunodeficiency syndrome (AIDS; Aquired immune deficiency syndrome- AIDS). Only it can be recognized as an independent acquired immunodeficiency disease.
The history of the discovery of AIDS dates back to 1981, when the Center for Disease Control (USA, Atlanta) published a report by groups of doctors from New York and Los Angeles about an unusual disease registered in homosexual men. It was characterized by a severe form of pneumonia caused by the opportunistic fungus Pneumocystis carinii. Subsequent reports provided data on the expansion of the group of patients and provided data on the presence of immunodeficiency in them, associated with a sharp decrease in the content of CD4+ T-lymphocytes in the circulation, accompanied by the development of infectious processes that can be caused, in addition to pneumocystis, by other facultative pathogens. Some patients developed Kaposi's sarcoma, which was characterized by an aggressive course that was unusual for it. By the time these materials were published, 40% of the identified patients had died. It later turned out that the epidemic of the disease had already captured equatorial Africa, where the disease spreads mainly through heterosexual sexual contact. The international medical community not only recognized the existence of a new nosological form - “acquired immunodeficiency syndrome” ( Aquired Immunodeficiency Syndrome), But
And announced the beginning of a pandemic of this disease. Such a dramatic debut of AIDS attracted universal attention to it, going far beyond the professional environment. In medical science, especially in immunology, the problem of AIDS has significantly influenced the distribution of effort and funding in the development of scientific research. This was the first time that a disease associated with a predominant lesion of the immune system turned out to be so significant in scientific and social terms.
TO early 2007 date There were 43 million people infected with HIV, of which 25 million died, the annual increase in this number is 5 million, and the annual mortality rate is 3 million. 60% of those infected live in sub-Saharan Africa.
In 1983, almost simultaneously in France [L. Montagnier (L. Montagnier)]
And United States of America [R.S. Gallo ( R.C. Gallo)] was determined
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the viral nature of AIDS and its causative agent, HIV (human immunodeficiency virus, Human Immunodeficiency Virus - HIV). It belongs to retroviruses, i.e. viruses in which RNA serves as the carrier of hereditary information, and it is read with the participation of reverse transcriptase. This virus belongs to the subfamily of lentiviruses - slow-acting viruses causing diseases with long incubation period. The HIV genus includes the species HIV-1, which is the causative agent typical shape AIDS, and HIV-2, which differs from HIV-1 in details of its structure and pathogenic action, but is generally similar to it. HIV-2 causes a milder variant of the disease, mainly found in Africa. The information below applies primarily to HIV-1 (except where otherwise noted). There are 3 groups of HIV - M, O and N, divided into 34 subtypes.
The current accepted view is that HIV-1 originated from a chimpanzee virus in West Africa (most likely in Cameroon, an HIV-endemic country) around the 1930s. HIV-2 originated from the simian virus SIVsm. HIV-1 variants are unevenly distributed around the world. In developed Western countries, subtype B predominates, in central Europe and Russia - subtypes A, B and their recombinants. Other variants predominate in Africa and Asia, with all known HIV subtypes present in Cameroon.
Morphology, genes and proteins of the human immunodeficiency virus
The structure of HIV is shown in Fig. 4.46. The virus has a diameter of about 100 nm. It is surrounded by a shell from which mushroom-shaped
Shell
Nucleocapsid proteins and enzymes
Nucleocapsid
Rice. 4.46. Scheme of the structure of human immunodeficiency virus 1 (HIV-1)
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Rice. 4.47. Genome structure of human immunodeficiency virus 1 (HIV-1). The location of genes on two RNA molecules of the virus is indicated
spines, the outer part of which is formed by the envelope protein gp120, and the membrane-adjacent and transmembrane parts are formed by the gp41 protein. The spikes represent trimers of these molecules. These proteins are involved in the interaction between the virus and the host cell, and the latter’s immune response is directed mainly against them. Deeper is the matrix layer, which acts as a frame. The middle part of the virus is formed by a cone-shaped capsid, which contains genomic RNA. Nucleoproteins and enzymes are also localized here: reverse transcriptase (p66/p51), integrase (p31–32), protease (p10) and RNase (p15).
The genetic structure of HIV and the proteins encoded by its genes are presented in Fig. 4.47. In two molecules of single-stranded RNA with a total length of 9.2 kb, 9 genes encoding 15 HIV proteins are localized. Sequences encoding virus structures are limited at the 5' and 3' ends by long terminal repeats (LTR - Long terminal repeats), which perform regulatory functions. Structural and regulatory genes partially overlap. The main structural genes are 3 - gag, pol and env. The gag gene determines the formation of group-specific antigens of the core - nucleoid and matrix. The pol gene encodes DNA polymerase (reverse transcriptase) and other nucleotide proteins. The env gene encodes the formation of the envelope proteins mentioned above. In all cases, the primary gene product is processed, i.e. breaks down into smaller proteins. Regulatory genes are located between the pol and env genes (vif, vpr, vpu, vpx, rev, tat genes) and, in addition, occupy the 3’-terminal part of the genome (fragments of the tat and rev genes, nef gene). Proteins encoded by regulatory genes are important for the formation of the virion and its relationship with the cell. Of these, the most important proteins are tat, a transcription transactivator, and nef (27 kDa), its negative regulator. Defective nef protein is detected in HIV-infected “long-livers” who do not experience disease progression.
The most important for the immunology of HIV infection, diagnosis and development of approaches to AIDS immunotherapy are the envelope proteins gp120 and gp41. The env gene is associated with extremely high HIV variability. The gene contains 5 constant (C) and five variable (V) regions; in the latter, the amino acid sequence varies from one virus isolate to another by 30–90%. The V3 variable loop is especially important for immunogenicity. The frequency of mutations in the env gene is 10-4–10-5 events per genome per cycle, i.e. 2–3 orders of magnitude higher than the normal frequency of gene mutations. A significant part of the molecule is occupied by carbohydrate residues.
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Infection of cells with human immunodeficiency virus
The process of infection of human cells by HIV and its subsequent replication includes several stages. In the early phase life cycle The following phases can be distinguished:
– binding of HIV to the cell surface (reception);
– fusion of the membranes of the virus and the cell and penetration of the virus into the cell (fusion and “undressing”);
– start of reverse transcription; formation of a pre-integration complex;
– transport of the preintegration complex into the nucleoplasm;
– integration of the provirus into the cell genome.
TO The stages of the late phase of the HIV life cycle include:
– transcription of viral RNA on the matrix of integrated proviral DNA;
– export of viral RNA to the cytosol;
– translation of viral RNA, protein processing;
– assembly of a viral particle on a cell membrane;
– release of the newly formed virion.
The main entry points for infection are the mucous membranes of the genitourinary and digestive tract. The penetration of the virus into the body is greatly facilitated in the presence of damage to the mucous membrane, but infection is possible even in their absence. In this case, the virus is captured by the processes of dendritic cells that penetrate the lumen of the organ. In any case, dendritic cells are the first to interact with HIV. They transport the virus to the regional lymph node, where it infects CD4+ T cells through the interaction of dendritic cells with T lymphocytes during the presentation of antigens.
Reception of HIV is due to mutual recognition of the trimer of the virus gp120 protein and the membrane glycoprotein CD4 of the host cell. The regions responsible for their interaction are localized on both molecules. On the gp120 molecule, the indicated region is located in its C-terminal part (residues 420–469), in addition, there are 3 more regions important for the formation of the interaction site with CD4, and a region (254–274) responsible for the penetration of the virus into the cell after binding to membrane CD4. On the CD4 molecule, the binding site for gp120 is located in the N-terminal V domain (D1) and includes the sequences of residues 31–57 and 81–94.
Since the CD4 molecule serves as the receptor for HIV, the range of target cells of this virus is determined by its expression (Table 4.20). Naturally, its main targets are CD4+ T lymphocytes, as well as immature thymocytes expressing both coreceptors (CD4 and CD8). Dendritic cells and macrophages that weakly express CD4 on the membrane are also effectively infected with the virus and serve as its active producers (HIV replication in dendritic cells is even higher than in T lymphocytes). HIV targets also include other cells containing at least small amounts of CD4 on the surface - eosinophils, megakaryocytes, endothelial cells, some epithelial cells (thymic epithelium, intestinal M-cells) and nerve cells(neurons, microglial cells, astrocytes, oligodendrocytes), spermatozoa, chorioallantois cells, striated muscles.
680 Chapter 4. Immunity in protecting and damaging the body...
Table 4.20. State of immunological parameters in acquired immunodeficiency syndrome
Indicator | Preclinical | Clinical stage |
manifestations |
||
Lymphocyte count | ||
Normal or reduced | Less than 200 cells per |
|
1 µl blood |
||
Normal or increased | Normal or reduced |
|
(percentage may be |
||
CD4+ /CD8+ ratio | ||
Th1/Th2 ratio | Normal or reduced | |
Cytotoxicity activity | Promoted | |
ical T cells | ||
T cell response | Normal or reduced | Sharply depressed |
to mitogens | ||
Normal or reduced | ||
Antigenemia | Appears on | Absent |
2–8 weeks | ||
Antibodies in circulation | Usually appear after | Present |
Soluble factors in | Soluble forms of α-chain IL-2R, CD8, TNFR, |
|
circulation | β2-microglobulin, neopterin |
|
Function reduced |
||
Lymphoid tissues, asso- | Early decrease in content | Strong suppression |
ciated with mucus | CD4+ T cell reduction | T cells, especially subsets |
thick shells | CD4+ populations |
|
Innate immunity | Normal or depressed | Depressed |
Additional molecules necessary for the penetration of HIV into cells are its coreceptors - 2 chemokine receptors: CXCR4 (receptor for the chemokine CXCL12) and CCR5 (receptor for the chemokines CCL4 and CCL5). To a lesser extent, the role of coreceptor is inherent in more than a dozen chemokine receptors. CXCR4 serves as a coreceptor for HIV-1 strains cultured on T cell lines, and CCR5 is for strains cultured on macrophage lines (it is present on macrophages, dendritic cells, and also on CD4+ T cells). Both of these receptors are classified as rhodopsin-like, transmitting a signal into the cell through the G-protein associated with them (see section 4.1.1.2). Both chemoreceptors interact
With gp120 protein; the binding site for these receptors opens in the gp120 molecule after interaction with CD4 (Fig. 4.48). Different HIV isolates differ in their selectivity for certain coreceptors. Supportive role in reception HIV-2 is played by adhesion molecules, in particular LFA-1. When dendritic cells are infected in interaction
With HIV lectin receptor involved DC-SIGN.
4.7. Immunodeficiencies | |
Hypervariable regions of gp120
Rice. 4.48. Scheme of interaction between the virus and the target cell during its infection. Illustrated is one of the options for the interaction of T-cell receptor molecules and HIV-1 molecules, ensuring the penetration of the virus into the cell
Coreceptors play an important role in the fusion of the viral envelope with the cell membrane. On the viral side, the gp41 protein plays a major role in fusion. After the phases of fusion (fusion) and “undressing” of the virus, a reverse complex is formed, which provides reverse transcription with the formation of double-stranded proviral DNA.
With the help of the viral enzyme integrase, the cDNA is integrated into the cell's DNA, forming a provirus. The peculiarity of the integration of HIV genes into the cellular genome is that it does not require cell division. As a result of integration, a latent infection is formed, which usually involves memory T cells, “dormant” macrophages, which serve as a reserve of infection.
HIV replication occurs primarily or exclusively in activated cells. When CD4+ T cells are activated, the transcription factor NF-KB is induced, which binds to the promoters of both cellular and viral DNA. Cellular RNA polymerase transcribes viral RNA. The genes tat and rev are transcribed earlier than others, the products of which are involved in viral replication. Tat is a protein that interacts with long terminal sequences (LTR), which sharply increases the rate of viral transcription. Rev is a protein that promotes the exit of viral mRNA transcripts, both spliced and unspliced, from the nucleus. The viral mRNA released from the nucleus serves as a template for the synthesis of structural and regulatory proteins. The structural proteins gag, env, pol form a viral particle that buds from the cell.
Stimulation of lymphocytes with mitogens enhances HIV replication and its cytopathogenic effect. This may be facilitated by endogenous factors accompanying cell activation, induced in activated lymphocytes and macrophages (NF-κB has already been mentioned). Cytokines, especially TNFα and IL-6, may also be such factors. The first activates the transcription of HIV genes, the second stimulates the expression of HIV in host cells. Colony-stimulating factors GM-CSF and G-CSF have a similar effect. IL-1, IL-2, IL-3 and IFNγ can act as cofactors for HIV activation. Glucocorticoid hormones of the adrenal glands contribute to the implementation of the genetic program of HIV. IL-4, IL-7 and IFNα have opposite effects.
Immune response to HIV antigens
Acute viral infection is characterized by the relatively rapid formation of antigen-specific CD4+ and CD8+ T cells that synthesize IFNγ. This leads to a rapid drop in the virus content in the blood, but not its disappearance. The cellular response to HIV infection consists of the formation of antigen-specific CD4+ T helper cells and CD8+ T killer cells. Cytotoxic CD8+ T cells are detected throughout the entire course of AIDS, with the exception of late stages, while virus-specific CD4+ T cells are detected only in the early stages of the disease. CD8+ killer T cells kill infected cells before the virus leaves the cell, thereby interrupting viral replication. There is a clear inverse relationship between the titer of the virus in the blood plasma and the number of specific CD8+ T killer cells. Increased proliferative activity of CD4+ and CD8+ antigen-specific T cells correlates with slower disease progression. Patients containing a large number of CD8+ killer T cells are characterized by slow progression of the disease. CD4+ T cells also play an important role in viral clearance: there is a relationship between the proliferative response of CD4+ T cells to HIV antigens and plasma virus levels. It was noted that the severity of viremia is more closely inversely correlated with the production of IL-2 than IFNγ. During chronic viral infection, effector T cells are quantitatively preserved, but they change functionally. The ability of CD4+ T cells to synthesize IL-2 decreases; the formation of cytotoxic molecules by CD8+ T cells is weakened. The proliferative activity of CD8+ T cells decreases, believed to be a result of decreased production of IL-2 by CD4+ helper cells. The weakening of antiviral protection is facilitated by the differentiation of CD4+ T cells into Th2-type helpers. Even the spectrum of cytokines synthesized by CD8+ cytotoxic T lymphocytes is characterized by a predominance of Th2 cytokines.
It would be natural to expect that immune processes, which, albeit in a weakened form, develop in response to an invading virus, will be able to at least small degree protect the body from infection. In fact, if this happens, it will only be initial period diseases. Subsequently, despite the presence of antigen-specific CD4+ and CD8+ T cells, intensive replication of the virus occurs. This is a consequence of the selection of viruses with changes in epitopes recognized
Conditions caused by impaired cellular immunity (T cell defect) are severe combined immunodeficiency syndromes. In some patients, these forms of immunodeficiency can cause the development of extremely dangerous diseases (even life-threatening), while in others - only minor health problems. Let us dwell in more detail on diseases that develop when cellular immunity is impaired.
Chronic candidiasis of the skin and mucous membranes
Candidiasis (thrush) develops when the skin and mucous membranes are damaged by a fungal infection. In rare cases, the infection can spread to internal organs.Predisposition to the development of candidiasis exists with selective T-cell deficiency. Treatment of candidiasis requires the use of special antifungal drugs(some patients have to undergo lifelong maintenance therapy).
Metaphyseal chondrodysplasia
This disease is an autosomal recessive immunodeficiency disorder. Common in consanguineous marriages. Patients suffering from metaphyseal chondrodysplasia have fine, brittle hair and are highly susceptible to viral infections. In some cases, the disease can be cured by bone marrow transplantation.X-linked lymphoproliferative syndrome
X-linked lymphoproliferative syndrome is characterized by increased vulnerability to Epstein-Barr virus. Epstein-Barr virus can cause the development of dangerous diseases (infectious mononucleosis, aplastic anemia, cancer of the lymph nodes, chicken pox, vasculitis, herpes).It is worth noting that this disease is inherited only by males.
