Giant gluteal lipoma presenting as a sciatic hernia.

BACKGROUND: Sciatic hernia is considered to be the rarest hernia of pelvic floor with less than one hundred reports published worldwide. Lipoma in the hernia sac is even more unique pathology with only few cases reported in the literature. We report a case of gluteal lipoma protruding into pelvis, displacing rectum with bladder and presenting as a sciatic hernia. CASE PRESENTATION: A 53-year-old male presented with an expanding, slightly reducible, right gluteal painful mass, back pain, dull pressure in lower abdomen and perianal region radiating to the right buttock, urgent urination and defecation. Lower back pain lasts for more than 7 years, other symptoms­6 months. No spinal pathology was found on X-ray. On examination patient seemed well nourished, BMI 29, abdomen was soft, without palpable masses or signs of peritonitis. Digital rectal examination showed no pathology. There was a reducible lump on the lateral side of right gluteus. Computer tomography (CT scan) demonstrated a large intra- and extra-pelvic fatty mass traversing the greater sciatic foramen. The tumor was surgically removed through lower middle laparotomy approach. Subsequent pathological examination revealed lipoma. The patient recovered uneventfully, was discharged 8 days later. MRI scan was advised following 1 year after the surgery. CONCLUSION: The presence of a gluteal mass should always suggest the possibility of a sciatic hernia.

Giant gluteal lipoma presenting as a sciatic hernia.

BACKGROUND: Sciatic hernia is considered to be the rarest hernia of pelvic floor with less than one hundred reports published worldwide. Lipoma in the hernia sac is even more unique pathology with only few cases reported in the literature. We report a case of gluteal lipoma protruding into pelvis, displacing rectum with bladder and presenting as a sciatic hernia. CASE PRESENTATION: A 53-year-old male presented with an expanding, slightly reducible, right gluteal painful mass, back pain, dull pressure in lower abdomen and perianal region radiating to the right buttock, urgent urination and defecation. Lower back pain lasts for more than 7 years, other symptoms-6 months. No spinal pathology was found on X-ray. On examination patient seemed well nourished, BMI 29, abdomen was soft, without palpable masses or signs of peritonitis. Digital rectal examination showed no pathology. There was a reducible lump on the lateral side of right gluteus. Computer tomography (CT scan) demonstrated a large intra- and extra-pelvic fatty mass traversing the greater sciatic foramen. The tumor was surgically removed through lower middle laparotomy approach. Subsequent pathological examination revealed lipoma. The patient recovered uneventfully, was discharged 8 days later. MRI scan was advised following 1 year after the surgery. CONCLUSION: The presence of a gluteal mass should always suggest the possibility of a sciatic hernia.

Toll-like receptor 4 D299G polymorphism in metabolic disorders: a meta-analysis.

The toll-like receptor 4 (TLR4) plays a key role in the activation of innate immune response participating in the recognition of lipopolysaccharides. Changes in the innate immune response are involved in the pathogenesis of some metabolic disorders such as metabolic syndrome and type 2 diabetes mellitus (Met-S and T2DM). It has been recently shown the role of gut microbiota in the perpetuation of both insulin resistance and low-grade chronic inflammation. Some studies have reported that TLR4 D299G polymorphism is associated with metabolic disorders, however results have been inconsistent. Two recent meta-analyses showed that D299G is associated with inflammatory bowel disease and gastrointestinal cancers risk, two pathological states in which the luminal microbial flora-host cells interaction may be implicated. We conducted a systemic review of the published data considering all eligible published studies (six studies with 1696 cases and 3388 controls for D299G) and a meta-analysis was performed to evaluate the association between TLR4 D299G polymorphism and the risk for metabolic disorders. Five studies were identified for T2DM: three corresponding to Caucasian populations and two to mixed populations. The remaining study analyzed Met-S in a Caucasian population. We observed a significant association between D299G polymorphism and metabolic disorders (T2DM and Met-S) risk (OR = 0.566, 95 % CI: 0.347-0.925, p = 0.023) particularly in Caucasians. No association was found in mixed population subgroup. Our meta-analysis identified that the AG/GG genotypes of D299G are associated with decreased metabolic disorders risk.

Common variants in SOCS7 gene predict obesity, disturbances in lipid metabolism and insulin resistance.

BACKGROUND AND AIMS: Specific Suppressor of Cytokine Signaling (SOCS) members, such as SOCS7, may play a role in the development of insulin resistance (IR) owing to their ability to inhibit insulin signaling pathways. The objective was to explore the association between common variants and related haplotypes in SOCS7 gene and metabolic traits related to obesity, lipid metabolism and IR. METHODS AND RESULTS: 780 unrelated men were included in a cross-sectional study. We selected three tagged SNPs that capture 100% of SNPs with minor allele frequency ≥ 0.10. Analyses were done separately for each SNP and followed up by haplotype analysis. rs8074124C was associated with both obesity (p = 0.005) and abdominal obesity (p = 0.002) and allele C carriers showed, in comparison with TT carriers, lower BMI (p = 0.001) and waist circumference (p = 0.001). rs8074124CC- carriers showed lower fasting insulin (p = 0.017) and HOMA-IR (p = 0.018) than allele T carriers. rs12051836C was associated with hypertriglyceridemia (p = 0.009) and hypertriglyceridemic waist (p = 0.006). rs12051836CC- carriers showed lower fasting insulin (p = 0.043) and HOMA-IR (p = 0.042). Haplotype-based association analysis (rs8074124 and rs12051836 in that order) showed associations with lipid and obesity -related phenotypes, consistent with single locus analysis. Haplotype analysis also revealed association between haplotype CT and both decreased HDL-C (p = 0.026) and HDL-C (p = 0.014) as a continuous variable. CONCLUSIONS: We found, for the first time, significant associations between SOCS7 common variants and related haplotypes and obesity, IR and lipid metabolism disorders.

Retrorectal tumours: literature review and vilnius university hospital "santariskiu klinikos" experience of 14 cases.

OBJECTIVE: Retrorectal tumours are rare lesions in adults. The diagnosis of retrorectal lesion is often difficult and misdiagnosis is common. We present significant number of cases in view of scarce information available on this matter. METHODS: 14 patients were treated at Vilnius university hospital "Santariskiu klinikos" Centre of abdominal surgery from 1997 to 2010. The case notes of patients who underwent surgery for a retrorectal tumour were reviewed retrospectively. Surgical histories, operations, histological tumour type, surgical time, weight of the specimen, blood loss, length of stay were analysed. RESULTS: 13 patients underwent laparotomy, 1 patient had combined perineal approach and laparotomy. The most common types of the tumour were fibroma (3 cases), leiomyosarcoma (2 cases). 5 tumours (35,7%) were found to be malignant. 57% of the patients had undergone at least one operation prior to definitive treatment. 5 female patients were initially admitted under gynaecologists. Hospital stay varied from 14 days to 22 days (mean 16.2 days). A report of a representative case is presented. CONCLUSIONS: Retrorectal lesions in female patients can mimic gynaecological pathology. Patients with this rare pathology are to be treated in a major tertiary hospital by surgeons, who are able to operate safely in the retrorectal space.

Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition.

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.

Combined measurement of diabetes mellitus immunological markers: an assessment of its benefits in adult-onset patients.

The convenience of combining the measurement of antibodies to glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA-2A), and autoantibodies to insulin (IAA) in diabetic patients was assessed. We analysed 71 type 1 and 115 adult-onset diabetic patients. The latter were grouped into three categories according to the time of evolution to insulin dependence. The main findings were as follows: (i) in type 1 diabetes, the combined analysis of GADA and IA-2A showed a sensitivity of 87.4% and was not appreciably improved by adding IAA; (ii) out of 31 adults who required insulin immediately or within the first two years of diagnosis, 41.9, 29.0, and 6.5% were positive for at least one, two or all three, and all three markers, respectively; GADA was the most prevalent (35.5%) and IA-2A the least represented (16.1%); (iii) 34 adult patients with slow evolution to insulin dependence showed a completely different profile: 5.9% were GADA positive and 23.5% were IAA positive and no double or triple positivity was observed as all patients were IA-2A negative; and (iv) 50 type 2 patients who had not required insulin treatment showed a low incidence of GADA (4%) as the only marker present. We conclude that a combined double-antigen test for GADA and IA-2A is a useful strategy for prospective screening of type 1 diabetes. However, in adults, the profile of individual markers discloses the course to insulin dependence. Therefore, it seems advisable to measure the markers separately, to allow a better classification of these patients, and help define their treatment.

Immunologic and genetic markers in insulin-dependent diabetes mellitus (type 1) in an Argentine population.

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, insulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1% for ICA, 36.7% for IAA, 74.6% for GADA and 63.4% for ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8%, similar to the ICA512A-GADA (87.3%) or ICA512A-GADA-IAA combination (91.1%). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4%) were positive for IAA and a single case (1.5%) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.

Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli.

Most insulin-dependent diabetes mellitus patients gen-erate conformational autoantibodies to the islet-cell 65-kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type-1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild-type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350-359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino-acid substitution: Met-161-->Thr. This change impeded the co-expression of a 48-kDa by-product from an internal translation site. Also, a second 58-kDa by-product was identified as a GAD65 C-terminal proteolytic fragment that co-purifies with thioredoxin-M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild-type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.

Evaluación de la presencia de autoanticuerpos anti-insulina en población general y otra con alta incidencia de los haplotipos HLA DR4 y DQW3 / Evaluation of the presence of anti-insulin autoantibodies in the general population and in a population with high incidence of haplotypes HLA DR4 y DQW3

La diabetes mellitus tipo 1 (insulinodependiente) es una enfermedad autoinmune en la cual, previo a sus manifestaciones clínicas, suelen detectarse marcadores tales como autoanticuerpos anti-insulina (IAA), anti-islote pancreático (ICA) y contra la enzima glutámico decarboxilasa (anti GAD) de las células insulares. Además, existen factores predisponentes para su padecimiento como ciertos haplotipos HLA. En este trabajo se determinó un valor de corte para los valores de IAA a partir del cual se consideró como positiva su presencia, evalúandose luego una población de personas no diabéticas, no obesas y sin antecedentes de diabetes. Paralelamente se estudiaron muestras de sangre obtenidas de dos poblaciones diferentes: una de 678 personas provenientes de la población general y otra formada por 196 personas procedentes de diferentes tribus de la Provincia de Formosa. También se determinó la correlación entre los IAA y el serotipo HLA. Los resultados indican que el 1.8 de la población presentó valores superiores a los obtenidos en la población control, porcentaje que se elevó al 7.11 en la población indígena, la cual posee una elevada frecuencia de los haplotipos HLA DR4 y DQW3. Además se observó que en la población general los valores menores de IAA correspondieron a los haplotipos DR1, DQW1, DR4 y DQW3, mientras que los mayores lo hicieron a los haplotipos DR4, DQW3 y DR7. Estos resultados, que conforman la mayor casuística argentina de IAA, permiten definir valores de corte útiles para su diagnóstico en nuestro medio y confirman en nuestra población la correlación descripta en otros países y poblaciones entre IAA

Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen.

Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.

Expression of properly folded human glutamate decarboxylase 65 as a fusion protein in Escherichia coli.

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.

Detection of glutamic acid decarboxylase (GAD) autoantibodies by indirect immunofluorescence using CHO cells expressing recombinant human GAD65.

The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.

A case of allergy to human insulin associated with high IgG/IgE ratio for specific antibodies.

A 37-year-old woman with a previous history of allergic rhinitis and bronchial asthma presented local and generalized allergic reactions to bovine, porcine and human insulins. She developed ketoacidosis, high insulin requirements and transient hypoglycemic episodes. Several desensitizing schedules were applied in order to induce tolerance to human insulin therapy. In addition, the following parameters of the humoral immune response were measured in different serum samples taken within 13 days after one of the anaphylactic episodes: insulin antibodies (binding range = 29.4-47.8%; cutoff = 2.6%), total IgE (range = 500-850 IU/ml; normal values = 3.7-269 IU/ml), specific IgE (range = 0.42-0.83 PRU/ml; class 2) and subclasses of specific IgG (IgG1 = 97.2 SDS; IgG2 = 41.1 SDS; IgG3 = 9.9 SDS; IgG4 = 0.3 SDS, on day 1). A binding capacity of 31.8 IU insulin/l obtained by Scatchard analysis was in agreement with episodes of elevated insulin requirements and hypoglycemia. A high anti-insulin IgG/IgE ratio, along with high levels of specific IgG1 antibodies, suggested that the latter antibodies could be involved in the development of anaphylactic episodes.

Solid versus liquid phase assays in detection of insulin antibodies. Influence of iodination site on labelled insulin binding.

On type 1 newly diagnosed and on insulin treated diabetic patients, anti-insulin autoantibodies (IAA) and antibodies (IA) having the same specificity are respectively induced. Such immune response may be evaluated either by radiobinding assay (RBA) or enzyme-linked immunosorbent assay (ELISA). Both methodologies have been compared at previous International Workshops, which pointed out discrepancies in results. In this work, IAA/IA prevalence was assessed by displacement RBA and ELISA, in normal subjects, type 2 (treated with hypoglycaemic agents), insulin treated and newly diagnosed type 1 diabetic patients. Results showed a lack of RBA-ELISA agreement. An attempt was then made to determine whether such results were, at least in part, attributable to iodination site in Tyr-A14. For this purpose parallel RBA assays were carried out by using radiolabelled insulin at A14 and A19 Tyr residues. Control sera and samples from insulin treated and type 1 newly diagnosed diabetic patients were tested. Our results suggest that labelling position is not involved in artifactual binding of tracers, at least as a systematic phenomenon. In the majority of cases the variability in RBA-ELISA signal ratios are best explained in terms of differences in the basic principles operating in both methods instead of artifacts due to tracer preparation.

Heterologous humoral immune response in patients treated with human growth hormone from different sources.

The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [125I]hGH or [125I]bGH as tracers. Fifty-seven hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[125]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such a fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response.

Detection of autoantibodies against hGH in sera of idiopathic hypopituitary children.

Using reliable displacement radiobinding assay (RBA) and ELISA, the existence of anti-human growth hormone autoantibodies (hGHAA) was confirmed in idiopathic hypopituitary patients with growth impairment. Six of 35 hypopituitary patients (17.1%) and 1/85 (1.2%) control children proved positive for hGHAA by RBA (>control mean + 3 SD). IgG isotype-hGHAA by ELISAIgG (> control mean + 3 SD) were positive for 6/34 (17.7%) and 3/85 (3.5% hypopituitary and control children, respectively. Due to an asymmetry to the right of the ELISAIgG distribution, an alternative cutoff based on a nonparametric method was obtained, and positive results for hypopituitary children increased to 10/34 (29.4%). Three of 34 hypopituitary patients but no control children were positive for hGHAA of IgM isotype. The hGHAA were detected in children with or without perinatal problems. These autoantibodies may represent markers of a major autoimmune process involving a portion of the anterior pituitary and may contribute to the development of hypopituitarism in over 15% of hypopituitary children.