Biased use of the IGHV4 family and evidence for antigen selection in Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas

Extranodal marginal zone lymphomas (EMZL) are the most common lymphomas in the ocular adnexa. The etiology and potential role for antigenic stimulation in these lymphomas are still controversial. We have examined IGHV gene usage and mutations in 67 Chlamydophila psittaci-negative ocular adnexal EMZL. Clonal IGHV gene sequences were identified in 43 tumors originating from the orbit (19), conjunctivae (18) and lacrimal gland (6). Forty four potentially functional clonal IGHV gene sequences were detected with overrepresentation of the IGHV4 family and IGHV4-34 gene. All but 3 sequences were mutated with the average percent homology to the germ line of 93.5±6.1. Multinomial model and Focused binomial test demonstrated evidence for positive and/or negative antigen selection in 59% of the potentially functional IGHV genes. Intraclonal variation was detected in 8 of 11 tumor specimens. Overall our findings demonstrate that C. psittaci-negative ocular adnexal EMZL exhibit biased usage of IGHV families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating B-cell receptor-mediated antigen stimulation in the pathogenesis of these lymphomas.     

Increased numbers of P63-positive/CD117-positive cells in advanced adenoid cystic carcinoma give a poorer prognosis

ABSTRACT: ObjectivesThis study consisted of two parts. One part was to analyze the survival rates of adenoid cystic carcinoma (ACC) in Chinese and explain the difference between our data and the literature. The other was to analyze the relationship between the expression of CD117 and the histological grade and the prognosis. METHODS: A retrospective study of 80 ACC patients was performed. Clinical data were collected, and p63, CD117 were detected by immunohistochemical staining. RESULTS: Eighty patients received follow-ups 3 to 216 months after initial diagnosis. ACC occurred in the lacrimal gland (26.3%, n = 21), nasal cavity and parasinus (33.8%, n = 27) and other sites (40.0%, n = 33). The 5-year and 10-year survival rates were 66.41% and 10.16%, respectively. Over expression of CD117 was detected in p63-negative cells in 94.3% of cases and in p63-positive cells in 45.8%. The expression of CD117 in p63-positive cells was significantly associated with the histological grade (P<0.001) and prognosis (P = 0.037) in patients in the advanced stage. CONCLUSIONS: ACC had a good 5-year survival but poor 10-year survival in Chinese, which differed from the occidental data. More p63+/CD117+ cells were associated with a higher histological grade and poorer outcome.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1701457278762097.     

Anti-Inflammatory Effects of Rebamipide Eyedrop Administration on Ocular Lesions in a Murine Model of Primary Sj?gren's Syndrome

Background

Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb), a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren''s syndrome (SS). However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown.

Methods and Finding

Reb eyedrops were administered to the murine model of SS aged 4–8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment.

Conclusion

Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces.     

Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

Anatomy of the major lacrimal gland of rhesus monkeys (Macaca mulatta)

The major lacrimal gland of rhesus monkeys is impalpable within the fatty connective tissue of the upper lateral quadrant of the orbit. Acini of the lacrimal glands are composed of both sparsely and heavily granulated cells that histochemically resemble serous acinar cells of the submandibular salivary gland. The cytoplasmic granules are strongly periodic acid-Schiff (PAS)-positive, and some are also stained by alcian blue for acidic mucosubstances. The lacrimal gland has a simple duct system of intralobular ducts and interlobular excretory ducts. Lymphocytes and plasma cells are common in the periductal stroma. Major lacrimal glands of rhesus monkeys are suitable for comparative and correlative studies of lacrimal and salivary diseases and radiation responses.     

Isolation and characterization of cancer stem cells from cervical cancer HeLa cells

Cervical cancer is one of the most common gynecologic malignancies and poses a serious health problem worldwide. Identification and characterization of cervical cancer stem cells may facilitate the development of novel strategies for the treatment of advanced and metastatic cervical cancer. Breast cancer-resistance protein (Bcrp1)-positive cells were selected from a population of parent HeLa cells using flow cytometry. The invasion capacity of Bcrp1-positive and -negative cells was analyzed with a Boyden chamber invasion test. The tumorigenicity of these cells was determined by in vivo transplantation in non-obesity diabetes/severe combined immunodeficiency (NOD/SCID) mice. The Bcrp1-positive subpopulation accounted for about 7% of the parent HeLa cell population. The proliferative capacity of the Bcrp1-positive cells was greater than that of the Bcrp1-negative cells (P < 0.05). In the invasion assay, the Bcrp1-positive cells demonstrated a greater invasive capacity through the artificial basement membrane than their Bcrp1-negative counterparts. Following transplantation of 10⁴ cells, only the Bcrp1-positive cells formed tumors in NOD/SCID mice. When 10⁵ or 10⁶ cells were transplanted, the tumor incidence and the tumor mass were greater in the Bcrp1-positive groups than those in the Bcrp1-negative groups (P < 0.05). The Bcrp1-positive subpopulation cervical cancer stem cells.     

Clinicopathological characteristics of immunoglobulin G4-related sialadenitis

IntroductionImmunoglobulin G4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition. Forty-two cases with immunoglobulin G4-related sialadenitis (IgG4-RS) confirmed by histopathological and immunohistochemical assessment were studied to clarify the clinicopathologic characteristics of the salivary glands involved in IgG4-RS, especially the relationship between the histopathologic features and function of salivary glands or serum levels of IgG4.MethodsClinical, serologic, imaging and histopathological data of these cases were analyzed. CT volumes of submandibular, parotid, and lacrimal glands were calculated. The saliva flow rate was measured. Scintigraphy with 99mTc-pertechnetate was undertaken in 31 cases, and the concentration index (CI) and secretion index (SI) was calculated. Relationships between fibrosis severity and salivary gland function or serum IgG4 levels were analyzed.ResultsThe first symptom was swelling of bilateral submandibular or lacrimal glands. Physical examination showed multiple bilateral major salivary glands (including sublingual and accessory parotid glands) and lacrimal glands were enlarged in IgG4 RS. Multiple enlarged cervical lymph nodes were noted in 30 patients. Saliva flow at rest was lower than normal in 34 cases; stimulated saliva flow was lower than normal in 15 cases. Secretory function was reduced more severely in the submandibular glands than in the parotid glands. Serum levels of IgG4 were elevated in 95.2% of cases and 78.6% patients had increased IgE levels. Serum IgG4 level was higher and saliva secretion lower as glandular fibrosis increased.ConclusionsProminent changes in the morphology, histology, immunohistochemistry and secretion of the major salivary glands of IgG4-RS patients were accompanied by involvement of the lacrimal glands and cervical lymph nodes. Elevated IgE, allergic history, eosinophil infiltration suggest allergic reactions as a potential pathogenesis of IgG4-RS. Severity of glandular fibrosis correlated with salivary function and serum levels of IgG4.     

Dynamic Ocular Surface and Lacrimal Gland Changes Induced in Experimental Murine Dry Eye

Dry eye disease can be a consequence of lacrimal gland insufficiency in Sjögren’s Syndrome or increased tear film evaporation despite normal lacrimal gland function. To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period. Dry eye was induced in 6-week-old female C57BL/6 mice by exposing them to an Intelligently Controlled Environmental System (ICES). Sixty mice were housed in ICES for 1, 2, 4 and 6 weeks respectively. Twelve were raised in normal environment and received subcutaneous injections of scopolamine hydrobromide (SCOP) 3 times daily for 5 days. Another sixty mice were housed in a normal environment and received no treatment. Corneal fluorescein staining along with corneal MMP-9 and caspase-3 level measurements were performed in parallel with the TUNEL assay. Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs). Immunohistochemistry of excised LGs along with flow cytometry in cervical lymph nodes evaluated immune cell infiltration. Light and transmission electron microscopy studies evaluated LGs cytoarchitectural changes. ICES induced corneal epithelial destruction and apoptosis peaked at 2 weeks and kept stable in the following 4 weeks. In the ICES group, lacrimal gland proinflammatory cytokine level increases were much lower than those in the SCOP group. In accord with the lower proinflammatory cytokine levels, in the ICES group, lacrimal gland cytosolic vesicular density and size exceeded that in the SCOP group. ICES and SCOP induced murine dry eye effects became progressively more severe over a two week period. Subsequently, the disease process stabilized for the next four weeks. ICES induced local effects in the ocular surface, but failed to elicit lacrimal gland inflammation and cytoarchitectural changes, which accounts for less dry eye severity in the ICES model than that in the SCOP model.     

Establishing human lacrimal gland cultures with secretory function

Purpose

Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in–vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures.

Methods

Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA.

Results

Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15–20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed ‘spherules’ with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons).

Conclusion

The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future.     

大鼠泪腺睾酮受体直空负压ABC法光镜观察与免疫金探针超微结构定位

动物实验发现睾酮能改善干眼病动物模型干眼症状,促进泪腺分泌,但作用机理不明,本研究探讨泪腺细胞中是否存在睾酮受体。雌雄各8只大鼠的泪腺分别制成石蜡切片和超薄切片,睾酮分别采用ABC法及免疫金标记,通过真空负压ABC法光镜观察及免疫金探针超微结构定位进行细胞睾酮受体检查。结果：光镜下泪腺导管上皮细胞的胞浆及胞核中出现免疫染色阳性,泪腺上皮细胞则很少见到;电镜下泪腺细胞胞浆及核中可见金颗粒,对照组则染色阴性。结论 泪腺细胞存在着睾酮受体,睾酮通过受体对泪腺发生作用。     

Selenoprotein P Controls Oxidative Stress in Cornea

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.     

Accumulation of Secretory Vesicles in the Lacrimal Gland Epithelia Is Related to Non-Sjögren's Type Dry Eye in Visual Display Terminal Users

Previous observations in a rat model of a non-Sjögren''s syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.     

Hormonal influence on the secretory immune system of the eye: endocrine impact on the lacrimal gland accumulation and secretion of IgA and IgG

The objective of the current investigation was to explore the processes underlying the androgen control of tear IgA and to determine whether hormone exposure also modifies tear IgG content. In addition, studies evaluated the impact of diabetes on the androgen regulation of secretory immunity in the eye. Tears and lacrimal glands were collected from age-matched, adult male rats, which had undergone hypophysectomy, selective ablation of the anterior pituitary, streptozotocin-induced diabetes, sham-surgery and/or orchiectomy and had been exposed to vehicle or physiological amounts of testosterone for varying periods of time. Our findings demonstrated that testosterone administration selectively increased the accumulation of IgA, but not IgG, in tears and lacrimal glands of orchiectomized rats. This hormone effect was associated with a 2-fold enhancement of the IgA transfer from lacrimal tissue to tears; IgA movement was against a gradient. In contrast, androgen exposure had no significant influence on the lacrimal gland/tear transfer of IgG, which was down a 90-fold gradient. Testosterone action on the lacrimal gland appeared to involve an increase in IgA production, but not a consistent alteration in the total number of IgA-containing cells. Similarly, androgen exposure had no impact on the population of IgG-containing lymphocytes in lacrimal tissue. Of interest, ablation of the anterior or entire pituitary in orchiectomized rats, which procedure inhibits testosterone-induced stimulation of tear IgA levels, significantly reduced the total number of IgA-containing cells in the lacrimal gland. Induction of diabetes by streptozotocin injection to orchiectomized rats resulted in diminished tear IgA content and decreased numbers of lacrimal IgA-positive lymphocytes, but did not prevent the testosterone-associated rise in IgA antibody content. In summary, our findings demonstrate that androgens increase the lacrimal gland production and secretion of IgA, but not IgG.     

兔附件扭转模型的建立及其意义探讨

目的 建立一种操作简单、病变典型、稳定性好的兔在体附件扭转模型并探讨保留卵巢术后卵巢的病理变化和iNOS的改变.方法 40只日本大耳白兔采用随机数字法分为附件扭转(adnexal torsion,AT)模型组(n=32)和对照组(n=8).模型组兔将左侧附件按顺时针方向扭转3周,避开血管,以4/0丝线横穿扭转形成的3个螺旋圈后固定于左侧腹壁,然后再将之分成4组,每组8只,分别于扭转24、48、72、96h后解除扭转后取双侧卵巢;对照组假手术后96h后取双侧卵巢.所有切除之右侧卵巢组织行病理学研究的内对照,左侧卵巢取1/2行病理检测,另1/2行iNOS生化水平检测.结果 左侧附件扭转3周固定左侧腹壁24 h后可见卵巢充血、炎细胞浸润、细胞水肿;48h见较多的炎细胞浸润,细胞结构紊乱;72 h见大量炎细胞浸润,结构损坏和局灶性坏死;96 h见弥漫性细胞坏死;卵巢组织病理评分呈现相同的时相性变化.各组iNOS生化检测水平(左侧vs.右侧),AT组[24 h(3.542±0.987) vs.(1.521±0.214) U/mgprot,P＜0.01;48 h(4.986±1.321) vs.(1.832±0.321) U/mgprot,P＜0.01;72 h(7.991±1.832) vs.(1.124±0.357)U/mgprot,P＜0.01;96 h(6.991±1.227) vs.(1.732±0.572)U/mgprot,P＜0.01].且AT组卵巢均有iNOS不同程度表达,72 h组表达达高峰,96 h下降.结论 成功地制备兔附件扭转模型,方法简单、病变典型、重复性好,可模拟女性附件扭转的病理生理过程,对进一步研究附件扭转具有重要意义.初步认为附件扭转72h后卵巢发生不可逆改变,是临床处理附件扭转并保留卵巢的时间临界点.     

The genesis of Thy-1-lymphomas in NFS mice exposed to X irradiation

The kinetics of the appearance of potentially leukemic cells (PoLCs) for radiation-induced lymphoma in NFS mice was investigated by the opposite sex (male----female) transplantation assay. The origin of the cells of the lymphomas that developed in the host was decided by sex chromosome markers. The bone marrow and the spleen cells collected from mice 30 days after fractionated irradiation (1.7 Gy X 4) gave rise, upon transfer to 4-Gy-irradiated hosts, to tumors of either donor or host origin. Most tumors of donor origin were thymine-1-negative (Thy-1-) and surface immunoglobulin negative and classified as nonthymic lymphoma, while the tumors of host origin were mainly Thy-1-positive thymic lymphoma. In contrast, neither the bone marrow nor the thymus contained any PoLCs for thymic lymphoma 30 days after split-dose irradiation. These results indicate that PoLCs for Thy-1-lymphoma were induced in the bone marrow and spleens of NFS mice by the split-dose regimen which developed exclusively T-cell lymphomas in the absence of cell grafting.     

HE4、NF-κBp65和MMP-9在卵巢上皮性肿瘤中的表达及其临床意义

目的探讨HE4、NF-κBp65以及MMP-9的表达与上皮性卵巢肿瘤临床病理生物学行为的关系以及对患者预后的影响。方法应用免疫组化对80例卵巢上皮性癌、10例交界性上皮性肿瘤及10例良性上皮性肿瘤组织进行HE4、NF-κB p65及MMP-9蛋白的检测。结果 HE4、NF-κBp65及MMP-9蛋白的阳性表达率在卵巢癌组均高于交界性及良性肿瘤组（P〈0.05）。三个指标的表达与EOC的组织学分级、腹腔脏器及淋巴结转移以及PTNM临床分期有关（P〈0.05）;在EOC中,NF-κBp65分别与HE4、MMP-9的表达呈正相关（r1=0.673,P〈0.05;r2=0.775,P〈0.05）。多因素分析,PTNM分期、MMP-9的表达是影响卵巢癌术后患者预后的独立因素（P〈0.05）;HE4阳性组与阴性组5年生存率分别为12.7%和84.0%,MMP-9阳性组与阴性组5年生存率分别为8.7%和70.6%,差异均有统计学意义（P〈0.05）。结论 NF-κBp65可能通过上调HE4、MMP-9的表达促进EOC的浸润和转移,联合检测HE4、NF-κBp65以及MMP-9的表达可能作为预测和评价患者预后的生物学指标。     

A Comparison of the Effects of Estrogen and Cimicifuga racemosa on the Lacrimal Gland and Submandibular Gland in Ovariectomized Rats

This study aims to observe the effects of estradiol and Cimicifuga racemosa on the lacrimal gland and submandibular gland of ovariectomized rats. We randomly divided 20 adult female SD rats into four groups—a sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estradiol (OVX+ E), and ovariectomized group treated with the isopropanolic extract of Cimicifuga racemosa (OVX+ iCR). The SHAM group and OVX group used distilled water to instead the drugs. Two weeks after ovariectomy, the estradiol and iCR were administered for 4 weeks. Next, we used H&E staining and electron microscopy to observe any histological changes in the lacrimal and submandibular glands and immunohistochemical staining to observe the expressions of cleaved caspase-3 (Casp-3) and Cu-Zn SOD (superoxide dismutase). The H&E staining find that both drugs can prevent the cells of area from shrinkage in the two kinds of gland. But under the electron microscopy, estradiol and iCR have different efficacy. Estradiol is more effective at protecting mitochondria in lacrimal gland acinar cells than iCR, and iCR is more effective at suppressing endoplasmic reticulum expansion than estradiol. Both estradiol and iCR have a similar protective function on mitochondria in the submandibular gland. The protective function of the two glands may inhibit apoptosis by suppressing the expression of Casp-3. In addition, iCR increases the expression of Cu-Zn SOD in duct system of submandibular gland. The results suggest that both estradiol and iCR confer a protective effect on the lacrimal and submandibular glands of ovariectomized rats via different mechanisms.     

The HLA-DRB4 gene is present in half of the Spanish HLA-DQ2-negative celiac patients

We studied nine consecutive DQ2-negative celiacs [from a group of 186 consecutive celiac disease (CD) patients] for the presence of the HLA-DQB1, DRB1, and DRBx alleles. HLA-DR53 was present in only 5 out of 9 (55%) of DQ2-negative patients. DRB4 (DR53) positivity -39% of chromosomes--among Spanish DQ2-negative CD patients is due to both DR4- and DR7-positive cases. Spanish DQ2-positive patients show a high frequency of DR7/DR11 heterozygous carriers of DRB4 (DR53). One-third of our DQ2-negative celiac patients have DRB1*04 (DR4). Six patients are DR4 negative: at least one of the DQ2 alleles (DQA1*0501/DQB1*02) is present in four cases, but none of the alleles of risk, including DR53, were found in the remaining two cases, both of whom carry DQB1*06 alleles (*0602/3 and 0604). The fact that half of our DQ2-negative patients lack DRB4 (DR53) leads us to believe that this gene is not an essential factor to confer CD susceptibility.