Consensus Middle East and North Africa Registry on Inborn Errors of Immunity.

BACKGROUND: Inborn errors of immunity (IEIs) are a heterogeneous group of genetic defects of immunity, which cause high rates of morbidity and mortality mainly among children due to infectious and non-infectious complications. The IEI burden has been critically underestimated in countries from middle- and low-income regions and the majority of patients with IEI in these regions lack a molecular diagnosis. METHODS: We analyzed the clinical, immunologic, and genetic data of IEI patients from 22 countries in the Middle East and North Africa (MENA) region. The data was collected from national registries and diverse databases such as the Asian Pacific Society for Immunodeficiencies (APSID) registry, African Society for Immunodeficiencies (ASID) registry, Jeffrey Modell Foundation (JMF) registry, J Project centers, and International Consortium on Immune Deficiency (ICID) centers. RESULTS: We identified 17,120 patients with IEI, among which females represented 39.4%. Parental consanguinity was present in 60.5% of cases and 27.3% of the patients were from families with a confirmed previous family history of IEI. The median age of patients at the onset of disease was 36 months and the median delay in diagnosis was 41 months. The rate of registered IEI patients ranges between 0.02 and 7.58 per 100,000 population, and the lowest rates were in countries with the highest rates of disability-adjusted life years (DALY) and death rates for children. Predominantly antibody deficiencies were the most frequent IEI entities diagnosed in 41.2% of the cohort. Among 5871 patients genetically evaluated, the diagnostic yield was 83% with the majority (65.2%) having autosomal recessive defects. The mortality rate was the highest in patients with non-syndromic combined immunodeficiency (51.7%, median age: 3.5 years) and particularly in patients with mutations in specific genes associated with this phenotype (RFXANK, RAG1, and IL2RG). CONCLUSIONS: This comprehensive registry highlights the importance of a detailed investigation of IEI patients in the MENA region. The high yield of genetic diagnosis of IEI in this region has important implications for prevention, prognosis, treatment, and resource allocation.

Clinical features of Candidiasis in patients with inherited interleukin 12 receptor ß1 deficiency.

BACKGROUND: Interleukin 12Rß1 (IL-12Rß1)-deficient patients are prone to clinical disease caused by mycobacteria, Salmonella, and other intramacrophagic pathogens, probably because of impaired interleukin 12-dependent interferon γ production. About 25% of patients also display mucocutaneous candidiasis, probably owing to impaired interleukin 23-dependent interleukin 17 immunity. The clinical features and outcome of candidiasis in these patients have not been described before, to our knowledge. We report here the clinical signs of candidiasis in 35 patients with IL-12Rß1 deficiency. RESULTS: Most (n = 71) of the 76 episodes of candidiasis were mucocutaneous. Isolated oropharyngeal candidiasis (OPC) was the most common presentation (59 episodes, 34 patients) and was recurrent or persistent in 26 patients. Esophageal candidiasis (n = 7) was associated with proven OPC in 2 episodes, and cutaneous candidiasis (n = 2) with OPC in 1 patient, whereas isolated vulvovaginal candidiasis (VVC; n = 3) was not. Five episodes of proven invasive candidiasis were documented in 4 patients; 1 of these episodes was community acquired in the absence of any other comorbid condition. The first episode of candidiasis occurred earlier in life (median age±standard deviation, 1.5 ± 7.87 years) than infections with environmental mycobacteria (4.29 ± 11.9 years), Mycobacterium tuberculosis (4 ± 3.12 years), or Salmonella species (4.58 ± 4.17 years) or other rare infections (3 ± 11.67 years). Candidiasis was the first documented infection in 19 of the 35 patients, despite the vaccination of 10 of these 19 patients with live bacille Calmette-Guérin. CONCLUSIONS: Patients who are deficient in IL-12Rß1 may have candidiasis, usually mucocutaneous, which is frequently recurrent or persistent. Candidiasis may be the first clinical manifestation in these patients.

X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production.

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.

Successful treatment of Fusarium solani ecthyma gangrenosum in a patient affected by leukocyte adhesion deficiency type 1 with granulocytes transfusions.

BACKGROUND: Ecthyma gangrenosum (EG) manifests as a skin lesion affecting patients suffering extreme neutropenia and is commonly associated with Pseudomonas aeruginosa in immunocompromised patients. Leukocyte adhesion deficiency I (LAD I) which count among primary immunodeficiency syndromes of the innate immunity, is an autosomal recessive disorder characterized in its severe phenotype by a complete defect in CD18 expression on neutrophils, delayed cord separation, chronic skin ulcers mainly due to recurrent bacterial and fungal infections, leucocytosis with high numbers of circulating neutrophils and an accumulation of abnormally low number of neutrophils at sites of infection. CASE PRESENTATION: We report at our knowledge the first case of a child affected by LAD-1, who experienced during her disease course a multi-bacterial and fungal EG lesion caused by fusarium solani. Despite targeted antibiotics and anti-fungi therapy, the lesion extended for as long as 18 months and only massive granulocytes pockets transfusions in association with G-CSF had the capacity to cure this lesion. CONCLUSION: We propose that granulocytes pockets transfusions will be beneficial to heal EG especially in severely immunocompromised patients.

Omenn syndrome: two case reports.

Omenn syndrome is a variant of combined severe immunodeficiency due to mutations in RAG genes. It is characterized by polymorph symptoms and lethal outcome. We report on two cases of Omenn syndrome. Infants were aged 50 and 46 days. The clinical and biological signs were typical and complete in the first case. In the second case, only the cutaneous signs were present. Diagnosis was confirmed by genetic study. The Rag1 T631 mutation was found in these two patients. Hematopoietic stem cell transplantation could not be done and the evolution was fatal in both cases because of severe infectious episodes. Prenatal diagnosis was performed in the two families and each family has currently a healthy child. In conclusion, early diagnosis of Omenn syndrome may avoid infectious complications responsible for delay in therapeutic management. Genetic study confirms the diagnosis. The treatment usually consists of hematopoietic stem cell transplantation in association with immunosuppressive drugs. Prenatal diagnosis is very important to allow parents to have healthy children.

Inherited IL-12p40 deficiency: genetic, immunologic, and clinical features of 49 patients from 30 kindreds.

Autosomal recessive interleukin (IL)-12 p40 (IL-12p40) deficiency is a rare genetic etiology of mendelian susceptibility to mycobacterial disease (MSMD). We report the genetic, immunologic, and clinical features of 49 patients from 30 kindreds originating from 5 countries (India, Iran, Pakistan, Saudi Arabia, and Tunisia). There are only 9 different mutant alleles of the IL12B gene: 2 small insertions, 3 small deletions, 2 splice site mutations, and 1 large deletion, each causing a frameshift and leading to a premature stop codon, and 1 nonsense mutation. Four of these 9 variants are recurrent, affecting 25 of the 30 reported kindreds, due to founder effects in specific countries. All patients are homozygous and display complete IL-12p40 deficiency. As a result, the patients lack detectable IL-12p70 and IL-12p40 and have low levels of interferon gamma (IFN-γ). The clinical features are characterized by childhood onset of bacille Calmette-Guérin (attenuated Mycobacterium bovis strain) (BCG) and Salmonella infections, with recurrences of salmonellosis (36.4%) more common than recurrences of mycobacterial disease (25%). BCG vaccination led to BCG disease in 40 of the 41 patients vaccinated (97.5%). Multiple mycobacterial infections were rare, observed in only 3 patients, whereas the association of salmonellosis and mycobacteriosis was observed in 9 patients. A few other infections were diagnosed, including chronic mucocutaneous candidiasis (n = 3), nocardiosis (n = 2), and klebsiellosis (n = 1). IL-12p40 deficiency has a high but incomplete clinical penetrance, with 33.3% of genetically affected relatives of index cases showing no symptoms. However, the prognosis is poor, with mortality rates of up to 28.6%. Overall, the clinical phenotype of IL-12p40 deficiency closely resembles that of interleukin 12 receptor ß1 (IL-12Rß1) deficiency. In conclusion, IL-12p40 deficiency is more common than initially thought and should be considered worldwide in patients with MSMD and other intramacrophagic infectious diseases, salmonellosis in particular.

Characterization of a novel monoclonal antibody with restricted specificity to the free beta 2 integrin alpha M CD11b subunit.

Leukocyte cell surface expression and function of beta2 integrins require the intracellular association of alpha subunits, CD11a, b, c, d, respectively, with the common CD18 beta2 subunit. We have raised and characterized a murine MAb -- ME-MDF -- directed against the low affinity form of the human integrin alphaM subunit CD11b A-domain. MAb ME-MDF is an IgG2a that has a kDa of 2,45461 +/- 0.12 x 10(-9) M. MAb ME-MDF recognizes both the low and high affinity forms of the CD11b A-domain. Flow cytometry showed that ME-MDF does not recognize the heterodimeric CD11b/CD18 molecule at the surface of polymorphonuclear cells and the human monoblast cell line U937. Western blot analysis of U937 cell line cell surface proteins demonstrated that ME-MDF reacts specifically with the CD11b subunit but does not react with the heterodimeric CD11b/CD18 complex, a feature that differentiates it from other CD11b A-dom-specific MAbs. These observations suggest that ME-MDF recognizes an epitope that is involved in the association of the two subunits and hence is not accessible within the heterodimeric form of the CD11b/CD18 molecule. These data show that the CD11b A-dom engages not only the MIDAS but also the ME-MDF-specific epitope to associate with the CD18 subunit. We have also constructed, and expressed in the yeast Pichia pastoris, the corresponding recombinant scFv form of MAb ME-MDF and characterized the CDRs. MAb ME-MDF is characterized by short VH and VL CDR3. MAb ME-MDF and/or its recombinant scFv form would be very useful to study the structural basis of the association between the alpha and beta2 integrin subunits and to investigate the possibility of modulating CR3 cell surface expression by preventing subunit association.

Bone marrow transplantation without conditioning regimen in Omenn syndrome: a case report.

OS is a non-SCID immunodeficiency characterized by a poor outcome even after BMT. We report here a case of BMT without preparative conditioning regimen, and with a successful engraftment in a five-month-old infant with OS. The patient was transplanted with 15 x 10(8) bone marrow mononuclear cells/kg, from his HLA matched brother, without preparative regimen and GVHD prophylaxis. Immunological status was assessed before and after the BMT, and the engraftment was monitored with microchimerism analysis. Six days after BMT, an acute GVHD involving first the skin, then the liver and gut, complicated the post-transplantation course. An excellent engraftment was confirmed by donor chimerism over 95% respectively at day post-transplantation 30, 60, 90, and 150. The cellular immunity of the patient was restored, and infectious complications decreased after BMT. Later the patient experienced chronic GVHD, and he died on day post-transplantation 246 from GVHD. BMT without conditioning regimen for OS is feasible, but there must be a megadose cell transplantation, and appropriate prophylactic immunosuppressive treatment to prevent acute GVHD.

Enhanced patient serum immunoreactivity to recombinant Mycobacterium tuberculosis CFP32 produced in the yeast Pichia pastoris compared to Escherichia coli and its potential for serodiagnosis of tuberculosis.

CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (originally annotated as Rv0577 and also known as TB27.3) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced in Escherichia coli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris. Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals, by enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases (n = 40) from BCG vaccinees (n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli-expressed antigens. Overall, the trans-production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.

Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask.

Taguchi's methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or beta 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the alpha subunit CD11b. Anti beta 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi's methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways.