Lamina Lucida Research Articles

Full-thickness Skin Grafts for Hand Contractures in an Adult Patient with Junctional Epidermolysis Bullosa: A Case Report

Epidermolysis bullosa is a group of inherited skin fragility disorders with blister formation in the basement membrane zone. Chronic scarring after repeated blistering of the hands causes narrowing of the first web, flexion contractures of the digits, and pseudosyndactyly. Junctional epidermolysis bullosa is a type of epidermolysis bullosa that is characterized by blister formation in the lamina lucida. This condition is associated with survival into adulthood. In adult survivors, hand function might be required in order to participate in normal social activities. However, there is a lack of literature on the management of hand surgery for adult patients with junctional epidermolysis bullosa. We herein describe an adult patient with junctional epidermolysis bullosa who had pseudosyndactyly and flexion contractures in both hands. Five surgeries were performed. Contractures were released and reconstructed using split- or full-thickness skin grafts. Delayed wound healing was always observed due to epidermal necrosis in the graft. After epithelialization, a satisfactory functional outcome was obtained.

Mixed muco-cutaneous pemphigoid: Clinical and immunological features of 15 cases.

We describe a series of patients whose auto-immune bullous skin disease (AIBD) of the dermal-epidermal junction (DEJ) was characterized by clinical, immunological and ultrastructural features intermediate between bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP), and a recalcitrant course. From the database of the French reference centre for AIBD, we screened all the patients who were referred for an AIBD of the DEJ with a mucosal involvement, who neither met the diagnostic criteria for the diagnosis of BP, nor were typical of MMP. Sera were analysed by NC16A-ELISA and immunobloting against the C-terminal and LAD-1 parts of BP180. Skin biopsies were studied by direct immunoelectron microscopy (IEM). Fifteen patients (4 males, 11 females) of mean age 70.8 ± 11.8 years were included. The mucosal involvement was localized in oral cavity in all cases and in pharyngeal/laryngeal or genital area in 8 (53%), and 6 patients (40%), respectively. No patient had ocular involvement, nor atrophic or fibrosing scars. All patients had extensive skin lesions (mean BPDAI score =65.9 ± 24.4), which predominated on the upper body part. Direct IEM performed on 8 patients showed IgG deposits on the lamina lucida in all cases, and the lamina densa in 5 cases. All sera recognized NC16A, while none recognized BP-230 in ELISA. 10 out of the 13 tested sera (76.9%) contained IgG which recognized the C-terminal domain of BP180 and 10 sera (76.9%) the LAD-1 domain of BP180. Patients poorly responded to super potent topical corticosteroids and were treated with oral corticosteroids ± immunosuppressant in 13 cases (86.6%). This mixed muco-cutaneous pemphigoid differs from BP by the younger age of patients, multiple mucosae involvement, circulating antibodies against both the C- and N-terminal part of BP180, and very poor response to topical CS. It differs from MMP by extensive inflammatory skin lesions, absence of ocular involvement and atrophic/fibrosing scars.

Junctional epidermolysis bullosa: genotype-phenotype correlations

Junctional epidermolysis bullosa most commonly results from mutations in the LAMA3, LAMB3, LAMC2, COL17A1, ITGA6 and ITGB4 genes. Junctional epidermolysis bullosa is characterized by clinical heterogeneity. To date, scientific findings allow to evaluate correlations between the severity of clinical manifestations and genetic defects underlying in the development of the disease. A systematic literature search was performed using PubMed and RSCI, and keywords including junctional epidermolysis bullosa, laminin 332, collagen XVII, 64 integrin. The review includes description of clinical findings of junctional epidermolysis bullosa, mutation location and types, its impact on protein production and functions. To evaluate the impact of gene mutation on protein functions, this review explores the structure and functions of lamina lucida components, including laminin 332, collagen XVII and 64 integrin, which are frequently associated with the development of junctional epidermolysis bullosa. The correlation between severe types of junctional epidermolysis bullosa and mutations resulting in premature stop codon generation and complete absence of protein expression has been described. Although, genotype-phenotype correlations should be analyzed carefully due to mechanisms which enable to improve protein expression.

DYSTROPHIC EPIDERMOLYSIS BULLOSA: CASE REPORT

Epidermolysis bullosa is a rare disease in which blisters are observed on the skin or mucosa after minor trauma. This disease is classified according to the ultrastructural level of separation of the epidermis from the dermis, being the dystrophic variant characterized by the cleavage reaching the lamina lucida. A patient with a previous diagnosis of dystrophic epidermolysis bullosa presented for dental evaluation at a specialized dental care service. During the clinical examination, it was found that the patient had white spots on her upper teeth and regular oral hygiene, with swollen gums that showed bleeding when the stimulus was performed, in addition to supragingival calculus. The patient had several bullous lesions, characteristic of the condition, spread throughout the body. Scraping of the supragingival calculus and prophylaxis was performed; the procedures had to be performed slowly and carefully to avoid trauma and thus the appearance of bullous lesions.

LB969 Cicatricial junctional epidermolysis bullosa (CJEB): A forgotten phenotype

CJEB, a subtype of EB first described in 1985, is characterized by blistering with scarring that can result in a degree of pseudosyndactyly and scarring alopecia. Phenotypically it may resemble recessive dystrophic EB (RDEB) and be misdiagnosed as such; however on electron microscopy a split is seen at the level of the lamina lucida. We report 2 cases of patients with CJEB. Patient 1 is a 34yr old female with a homozygous in-frame deletion of exons 16 and 17 in COL17A1, and Patient 2 a 21yr old female with a homozygous nonsense mutation of LAMB3(c.1702C>T, p.Q568X).

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin β and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB. Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations. Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene. In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.

A Rare Skin Disorder with Bacteremia in a Neonate.

A Rare Skin Disorder with Bacteremia in a Neonate.

Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement.

Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone. To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant. Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing. Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant. Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance. A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.

Homozygous ITGA3 Missense Mutation in Adults in a Family with Syndromic Epidermolysis Bullosa (ILNEB) without Pulmonary Involvement

Homozygous ITGA3 Missense Mutation in Adults in a Family with Syndromic Epidermolysis Bullosa (ILNEB) without Pulmonary Involvement

Fundamentalin vitro3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments

Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmeticsin vitrotesting and for the development of complex skin wound therapeutics.In vitrothree-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamentalin vitrotool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection.

CLINICAL CASES OF BULLOUS EPIDERMOLYSIS IN NEWBORNS

Summary. Hereditary bullous epidermolysis (BE) is a group of genetically and clinically heterogeneous diseases characterized by the formation of blisters and erosion due to injury on the skin and mucous membranes. Different forms of BE can be accompanied by various extracutaneous complications, such as blisters and erosion on the cornea and mucous membranes, stenoses and strictures of the respiratory system, gastrointestinal tract, urinary system, muscular dystrophy, and malignant tumors.
 Hereditary bullous epidermolysis is divided into three types, depending on the level of blister formation: simple, borderline, and dystrophic. Simple BE is characterized by the stratification of epidermis due to the keratinocyte cytolysis. Borderline BE means that blisters are formed at the border of the epidermis and dermis due to the splitting of the lamina of the basement membrane (lamina lucida), while dystrophic BE has blisters that are formed under the dense plate of the basement membrane (lamina densa), which exfoliates the dermis.
 Currently, mutations have been identified in more than 10 genes encoding the structural proteins of keratinocytes and the basal membrane of the skin and mucous membranes. A common feature of these proteins is their involvement in the formation of strong bonds between the epithelium and the basement membrane. The nature of the mutations and their localization determine the severity of the clinical manifestations of BE. Mutation information is a prerequisite for effective medical and genetic counseling, prenatal and preimplantation DNA diagnosis.
 Therefore diagnosis and prescribing appropriate treatment and follow-up care is an important task for neonatologists and pediatric dermatologists. As manifestations of hereditary BE are numerous, a specialized center is required for optimal care, where multidisciplinary care will be provided (neonatologists, pediatric surgeons, pediatric dermatologists, etc.).
 The purpose of clinical observation is to pay attention of specialists to this rare disease, and to present 2 clinical cases of bullous epidermolysis in newborns who were admitted to the anesthesiology and intensive care unit of newborns of Vinnytsia Regional Children's Clinical Hospital almost at the same time.

Inherited epidermolysis bullosa: update on the clinical and genetic aspects

Inherited epidermolysis bullosa is a group of genetic diseases characterized by skin fragility and blistering on the skin and mucous membranes in response to minimal trauma. Epidermolysis bullosa is clinically and genetically very heterogeneous, being classified into four main types according to the layer of skin in which blistering occurs: epidermolysis bullosa simplex (intraepidermal), junctional epidermolysis bullosa (within the lamina lucida of the basement membrane), dystrophic epidermolysis bullosa (below the basement membrane), and Kindler epidermolysis bullosa (mixed skin cleavage pattern). Furthermore, epidermolysis bullosa is stratified into several subtypes, which consider the clinical characteristics, the distribution of the blisters, and the severity of cutaneous and extracutaneous signs. Pathogenic variants in at least 16 genes that encode proteins essential for the integrity and adhesion of skin layers have already been associated with different subtypes of epidermolysis bullosa. The marked heterogeneity of the disease, which includes phenotypes with a broad spectrum of severity and many causal genes, hinders its classification and diagnosis. For this reason, dermatologists and geneticists regularly review and update the classification criteria. This review aimed to update the state of the art on inherited epidermolysis bullosa, with a special focus on the associated clinical and genetic aspects, presenting data from the most recent reclassification consensus, published in 2020.

Eosinophils in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disorder with substantial morbidity and mortality. BP is regarded as a disorder driven by IgG due to BP180 and BP230 IgG autoantibodies, yet, new advances highlight the function of eosinophils and IgE autoantibodies in BP. Evidence supports that eosinophils are involved in BP pathogenesis, notably, these include the presence of IL-5, eotaxin, and eosinophil-colony stimulating factor in blister fluid. Peripheral blood eosinophilia is present in nearly 50% of affected patients, eosinophils are found against the dermo-epidermal junction (DEJ) when BP serum is present and metalloprotease-9 is secreted at blister sites. Blister fluid of BP patients contains eosinophil granule proteins which are located along the lamina lucida of the basement membrane zone (BMZ) in patients with BP and correspond with disease clinically, eosinophil extracellular traps (EET) have been linked to DEJ splitting, IL-5 activated eosinophils cause DEJ separation when BP serum is present, and eosinophils are requisite to drive anti-BP180 IgE mediated blistering of the skin. Yet, the mechanism whereby eosinophils contribute to the pathogenesis of BP remains to be explored. In this review, we examined the role of eosinophils in BP while offering a basis to explain the pathomechanisms of eosinophils in BP.

Pioneers in Dermatology and Venereology: an interview with Prof. Louis M.M. Dubertret

Pioneers in Dermatology and Venereology: an interview with Prof. Louis M.M. Dubertret

Autoantibody Profile of a Cohort of 54 Italian Patients with Linear IgA Bullous Dermatosis: LAD-1 Denoted as a Major Auto-antigen of the Lamina Lucida Subtype.

Linear immunoglobulin A (IgA) bullous dermatosis (LABD) is characterized by the presence of multiple IgA autoantibodies, and a comparatively lesser number of IgG antibodies, directed against different hemidesmosomal antigens. The main autoantigens are LAD-1, LABD-97, BP180 and BP230, type VII collagen and laminin 332. The serology of 54 Italian patients with LABD was studied retrospectively using enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and indirect immunofluorescence on monkey oesophagus and salt-split skin. Among these, indirect immunofluorescence of salt-split skin elicits the greatest sensitivity. Sixty-three percent of the sera were observed to be positive, with a lamina lucida pattern observed in 48%, a sub-lamina densa pattern in 2% and a mixed pattern in 13% of cases. IgA reactivity to LAD-1 on immunoblotting was found in 52% of sera, to BP180-NC16A by ELISA in 32% and to BP230 in 26%. Only 17% of patients had circulating IgG autoantibodies. LAD-1 was determined to be a major autoantigen of the lamina lucida subtype. Combined serological assays demonstrated a high sensitivity (82%), suggesting that this approach could support diagnosis when a biopsy is not feasible or direct immunofluorescence results are negative.

Role of Immunofluorescence antigen mapping in the diagnosis of Epidermolysis Bullosa - A rare genetic blistering disorder

Background: Epidermolysis Bullosa is a rare genetic mechanobullous skin and mucosal fragility with blistering. It is classified into: Epidermolysis (Simplex), lucidolytic (Junctional) and dermolytic (Dystrophic). The disease is extremely debilitating necessitating early detection and treatment. Immunofluorescence mapping is gold standard of diagnosis. Objective: To determine types and inheritance patterns of cutaneous and extracutaneous Epidermolysis Bullosa by immunofluorescence mapping. Methods: 5 µm frozen section from lesions /artificial bulla are collected over multispot PTEF coated slides. Incubation done with primary antihuman mouse antibodies (IgG) and with FITC conjugated anti-IgG mouse secondary antibodies. Antibodies used against Collagen IV, Collagen VII, cytokeratin 4, cytokeratin 14, Laminin 332, alpha6 and beta4 integrins. Healthy skin is positive control. Results: In Simplex type blister and split is above lamina densa with reduced CK14 at base (Dominant). Anti-integrins, laminin 332 and collagen IV all are present at base. In Junctional type, collagen IV present at base, cytokeratin or integrin at roof, and cleavage is at lamina lucida. Laminin 332 is reduced (dominant) or absent (recessive). In Dystrophic type blister is below lamina densa. Collagen IV or VII stains roof of blister. Collagen VII is absent in recessive type and reduced in dominant type. Limitations: Rare disease Conclusion: Agreement of clinical diagnosis and mapping is moderate indicating mapping is an essential adjunct.

Ultrastructural characterization of damage in the basement membrane of facial melasma.

The pathogenesis of melasma is not fully understood, and the role of skin basement membrane zone (BMZ) alterations in disease development and the maintenance of hypermelanogenesis are also poorly known. We performed a comparative study to characterize the ultrastructural alterations that occur in BMZ in melasma and adjacent normal skin, as well as we discuss the implications of these changes in the physiopathology of the disease. Pairs of facial skin biopsies (2 mm) from 10 women with melasma and normal skin (< 2 cm apart) were processed by Transmission Electronic Microscopy or immunohistochemistry for Melan-A counterstained with Periodic acid-Schiff stain. Cytoplasmic organelles (from keratinocyte or melanocyte), BMZ damage were assessed and melanocyte counting (total and pendulous) was done. There was greater amount of cytoplasmic organelles inside basal keratinocytes and melanocytes in melasma, as well as structural damaged areas in the lamina densa (disruptions, gaps, lower density and thinning) and anchoring fibrils (lamina lucida), compared to healthy adjacent skin. Areas with pendulous melanocytes are characterized by discontinuity of BMZ ultrastructure. The prominence of cytoplasmic organelles from melanocytes and keratinocytes evidences the involvement of both cell groups in melasma. The damage in the lamina densa and lamina lucida suggest the role of upper dermis injury/repair process in the pathogenesis of the disease.

Ultrastructural examination of basement membrane pathology in horses with insulin-induced laminitis

The third phalanx of the equine digit is suspended within the hoof capsule by a specialized interdigitating dermoepidermal layer called the lamellae, which fails during laminitis. Pathology of the basement membrane (BM), which interfaces epidermis and dermis, is evident during acute laminitis. However, BM damage appears to be less prevalent in ponies with the insulin-associated form of laminitis. The aim of the present study was to investigate changes to the ultrastructure and morphometry of the lamellar BM in the acute phase of insulin-induced laminitis in horses. Lamellar tissue from the left forefoot of 3 horses with acute hyperinsulinemic laminitis was examined with transmission electron microscopy and compared with tissue from normal horses. Lamellar BM width and hemidesmosome (HD) density were assessed every 5 μm along ∼200 μm of secondary epidermal lamellar BM. The BM zone of treated horses was extensively disorganized with loss of uniformity of the lamina lucida and lamina densa, fragmentation and disorientation of HDs, and cytoskeletal disengagement of the HDs. The mean (±SD) lamellar BM was twice as wide in treated (0.25 ± 0.05 μm), compared with control (0.14 ± 0.02 μm), horses. The HD density (HDs/μm) was reduced by half in the treatment group (1.88 ± 0.37), compared with controls (3.6 ± 0.13). The reduced number of HDs in horses with laminitis may contribute to the weakening of the dermoepidermal junction and lamellar failure. Disassembly of HDs during excessive cellular proliferation, secondary to hyperinsulinemia, may account for HD loss. Further investigation of the underlying etiopathogenesis of BM dysfunction during hyperinsulinemic laminitis in horses may facilitate an improved understanding of the disease.

Electron Microscopic and Immunohistochemical Findings of the Epidermal Basement Membrane in Two Families with Nail-patella Syndrome.

Nail-patella syndrome is an autosomal dominant disorder characterized by nail dysplasia and skeletal anomaly. Some patients have been shown to have ultrastructural abnormalities of the glomerular basement membrane that result in nephrosis. However, little has been reported on the epidermal basement membrane in this condition. This paper reports 2 families with nail-patella syndrome. Direct sequencing analysis of LMX1B revealed that family 1 and family 2 were heterozygous for the mutations c.140-1G>C and c.326+1G>C, respectively. To evaluate the epidermal basement membrane zone, ultrastructural and immunohistochemical analyses were performed using skin specimens obtained from the dorsal thumb. Electron microscopy showed intact hemidesmosomes, lamina lucida, lamina densa, and anchoring fibrils. Immunofluorescence studies with antibodies against components of the epidermal basement membrane zone revealed a normal expression pattern among the components, including type IV collagen. These data suggest that nail dysplasia in patients with nail-patella syndrome is not caused by structural abnormalities of the epidermal basement membrane.

Idiopathic linear IgA bullous dermatosis: prognostic factors based on a case series of 72 adults.

Linear IgA bullous dermatosis (LABD) is a clinically and immunologically heterogeneous, subepidermal, autoimmune bullous disease (AIBD), for which the long-term evolution is poorly described. To investigate the clinical and immunological characteristics, follow-up and prognostic factors of adult idiopathic LABD. This retrospective study, conducted in our AIBD referral centre, included adults, diagnosed between 1995 and 2012, with idiopathic LABD, defined as pure or predominant IgA deposits by direct immunofluorescence. Clinical, histological and immunological findings were collected from charts. Standard histology was systematically reviewed, and indirect immunofluorescence (IIF) on salt-split skin (SSS) and immunoblots (IBs) on amniotic membrane extracts using anti-IgA secondary antibodies were performed, when biopsies and sera obtained at diagnosis were available. Prognostic factors for complete remission (CR) were identified using univariate and multivariate analyses. Of the 72 patients included (median age 54 years), 60% had mucous membrane (MM) involvement. IgA IIF on SSS was positive for 21 of 35 patients tested; 15 had epidermal and dermal labellings. Immunoelectron microscopy performed on the biopsies of 31 patients labelled lamina lucida (LL) (26%), lamina densa (23%), anchoring-fibril zone (AFz) (19%) and LL+AFz (23%). Of the 34 IgA IBs, 22 were positive, mostly for LAD-1/LABD97 (44%) and full-length BP180 (33%). The median follow-up was 39 months. Overall, 24 patients (36%) achieved sustained CR, 19 (29%) relapsed and 35% had chronic disease. CR was significantly associated with age > 70 years or no MM involvement. No prognostic immunological factor was identified. Patients with LABD who are < 70 years old and have MM involvement are at risk for chronic evolution.