Hypermobility Assessment in 1,004 Adult Patients Presenting with Hip Pain: Correlation with Diagnoses and Demographics.

BACKGROUND: The strength of the association between hypermobility and developmental dysplasia of the hip (DDH) in adults is unknown. We sought to analyze this relationship in a prospective, blinded, institutional review board-approved, observational study. The hypothesis was that the prevalence of generalized joint hypermobility (GJH) would be significantly higher in patients with hip dysplasia than in those with other hip diagnoses on the basis of clinical observations of joint laxity. METHODS: One thousand and four consecutive new patients (390 males and 614 females) seen over a 4-year period were evaluated for hypermobility of the hip using 2 criteria: the Beighton 9-point physical examination criteria and the Hakim-Grahame 5-item history questionnaire. Diagnosis, age, sex, and race were tested as predictors of hypermobility. Patient-reported outcome scores from the International Hip Outcome Tool (iHOT-12) and the modified Harris hip score (mHHS) were also assessed. RESULTS: DDH was the primary diagnosis in 33.2% of the patient population. Patients who had dysplasia without osteoarthritis (OA) had a significantly elevated prevalence of GJH (77.9%) compared with those with nondysplastic hips (32.8%; p < 0.0001) or with patients who had dysplasia and OA (35.7%; p < 0.0001) according to either method. The odds ratio (OR) for patients with DDH versus those with other diagnoses was 7.1 (95% confidence interval [CI]: 5.1 to 10.0). The prevalence of hypermobility was significantly greater in females than in males (OR = 4.2 [95% CI: 3.2 to 5.5]; p < 0.0001). The prevalence of GJH was inversely proportional to age. There was a significantly reduced prevalence of GJH observed in Hispanic patients (p < 0.05) compared with other races. GJH was not a predictor of patient-reported outcome scores (p = 0.51 for iHOT-12 and p = 0.44 for mHHS). CONCLUSIONS: To our knowledge, this study is the first to establish a strong association between hypermobility and DDH in adults, confirming the hypothesis. We recommend utilizing both the Beighton and Hakim-Grahame scoring systems together as routine components of the history and physical examination for patients with hip dysplasia. Further research is warranted to explore the genetic basis and potential causal relationships between soft-tissue laxity and skeletal dysplasia, as well as improvements in assessment tools. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

[Expression and significance of miR-210 in the epididymis in rats with varicocele and following varicocelectomy].

Objective: To investigate the expression of microRNA 210 (miR-210) in the epididymis of rats with varicocele and changes in miR-210 expression following high spermatic vein ligation, so as to explore the significance of the surgery in treating varicocele. Methods: A total of 21 male Sprague-Dawley (SD) rats aged 7 weeks were randomly divided into control group (n=7), experimental group (n=7), and surgical group (n=7). Varicocele model was established in both the experimental and surgical groups, while only vein isolation was performed in the control group. After 8 weeks, spermatic vein diameter were measured in the control and experimental rats, and collected the left epididymis (fixed in formaldehyde and frozen in refrigerator at -80 ℃). In the surgical group, left high spermatic vein ligation was performed, and the left epididymis was collected after 4 weeks as in the control and the experimental groups. The fixed epididymis tissues were treated with HE staining for observation of tissue injuries. The miR-210 expression in the epididymis was detected with reverse transcription-polymerase chain reaction (RT-PCR). At last every group had 5 rats. Results: The pathological examination showed that the number and distribution of mature sperms in epididymal duct in the experimental group were lower and less even compared to the control group, while the two indicators in the surgical group were better than those in the experimental group. The diameter of the left spermatic vein in the experimental group and pre-treatment surgical group were significantly enlarged than in the control group (P<0.01). The expression of miR-210 in the left epididymis in the experimental group was significantly higher compared with the control group(1.32±0.06 vs 0.98±0.14, P<0.01), while the expression of miR-210 in the left epididymis in the surgical group was significantly decreased compared with the experimental group (0.96±0.16 vs 1.32±0.06, P<0.01); the difference between the control group and the surgical group was not statistically significant (P>0.05). Conclusion: The expression of miR-210 in the epididymis may be increased by varicocele and reduced after high ligation of the affected spermatic vein.

In-situ polymerase chain reaction: foundation of the technology and today's options.

"In situ PCR" is the marriage of two established technologies in molecular genetics, the polymerase chain reaction (PCR) and in situ hybridization (ISH). It is based on the amplification within intact cells or tissue sections of specific gene sequences, or mRNA species, to levels detectable by ISH and/or immunohistochemistry. Methods to achieve in situ PCR, while sharing fundamental steps, have differed between different laboratories. On the basis of our own experience, in situ PCR appears to be best suited for the detection of DNA in single cell preparations, in which fixation and pre-treatments can be optimally controlled. Emphasis is placed on the requirement for appropriate and meaningful controls at the multiple steps involved. It is instructive to the view the emergence of this new technology in perspective. In situ PCR has not developed in isolation and is just one of several creative approaches that have been employed in recent years to study nucleic acids (DNA and RNA) intracellularly. Some approaches are more suitable for detection of mRNA, or viral RNA, while others are more easily applied to chromosomal DNA. Some further techniques, such as the isothermal self-sustained sequence replication (3SR), refined in-situ transcription (PRINS), or high sensitivity histochemical detection systems, will complement or even add to the potential of situ PCR. It is highly probable that tests will emerge, based on investigation of unique genetic markers, with important roles in specialized diagnostic laboratories for the evaluation of viral diseases, as well as hematological and other malignancies.

Sensitization to cat allergen is associated with asthma in older men and predicts new-onset airway hyperresponsiveness. The Normative Aging Study.

To explore the relationship between sensitization to inhalant allergens and adult asthma, we performed two nested case-control studies of men being followed in the VA Normative Aging Study. In Study A, 46 subjects (mean age, 61.2 +/- 8.1 yr) with symptoms of asthma and an abnormal methacholine challenge test (cases) were compared with 92 age- and smoking-history-matched subjects, who denied symptoms and had normal methacholine challenge tests (controls). The age of onset of wheezing symptoms for the cases was 49.0 +/- 15.7 yr. Serum IgE reactivity to the aeroallergens Der P 1 and 2, cat, ragweed, and mouse was compared in cases and controls. Cases were more likely to be sensitized to cat allergen (23.9% versus 4.4%, p < 0.001) than were controls. Prevalences of sensitization to Der p 1, Der p 2, ragweed, and mouse were low and similar in the two groups. In Study B, 33 cases who developed new onset airway hyperresponsiveness on methacholine challenge testing were compared with 66 age-matched controls who maintained normal methacholine challenge tests. Cases had a higher prevalence of serum IgE reactivity to cat allergen (18.2% versus 6.1%, p = 0.059) and Der p 2 (21.2% versus 10.6%, p = 0.153) measured in serum obtained 3 yr before the development of airway hyperresponsiveness. These results suggest that in older men, sensitization to cat allergen is associated with asthma and that sensitization predates airway hyperresponsiveness to methacholine.

Treatment of cat allergy with T-cell reactive peptides.

We induced in allergic humans the counterpart of murine experimental T-cell tolerance. T-cell lines from cat-allergic humans were used to map T-cell epitopes for the principal allergen of cat dander, Fel d 1. Two peptides of 27 amino acids each were synthesized to contain the dominant epitopes (ALLERVAX CAT). After a safety trial, we carried out a blinded study of the dose required for efficacy. We randomly divided 95 cat-sensitive patients into placebo, 7.5 micrograms, 75 micrograms, and 750 micrograms groups. Patients received a subcutaneous injection weekly for 4 wk. Before and after treatment, patients were exposed in a room inhabited by live cats and scored by nose and lung symptoms. Baseline nasal and lung scores (+/-SEM) were 6.2 +/- 0.56 and 5.4 +/- 0.73 in the 750 micrograms group; 7.8 +/- 0.53 and 4.7 +/- 0.68 in the placebo group. Six weeks after treatment, scores adjusted for baseline differences were reduced in the 750 micrograms group: -2.3 +/- 4.9 and -2.3 +/- 0.59 compared with -0.84 +/- 0.50 and -0.85 +/- 0.62 in the placebo group. The 75 micrograms group showed intermediate effects and the 7.5 micrograms group no effect. Linear trend analysis indicated a significant dose response effect: p = 0.05 for nose and 0.03 for lung symptoms. Allergic side effects occurred an hour or more after the first 750 micrograms dose in 16 of 24 patients but required little or no treatment with one exception. T-cell reactive treatment peptides safely improved allergic responses to cats.

Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR.

To evaluate reliable methods for detection of hepatitis C virus (HCV) infection in routinely processed liver biopsies we analyzed formaldehyde-fixed and paraffin-embedded liver specimens of 10 patients with serological confirmed HCV infection. We compared (1) conventional histology; (2) indirect immunofluorescence using the mAb TORDJI-22 (Clonatec, Paris, France); (3) RT-PCR using total RNA and Southern blotting with chemiluminescent detection; (4) non-radioactive in-situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes; (5) direct in-situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and (6) indirect in-situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. [Gastroenerology 1993;104:595] together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies specimens are adequate; (2) the commercially available mAb TORDJI-22 appears to crossreact with non-HCV epitopes, resulting in false positives; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in-situ RT-PCR or ISH; and (5) direct in-situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. It is concluded, that several molecular methods for HCV detection must await further improvements of protocols to be suitable for routine diagnostics on paraffin-embedded liver biopsies.

In situ polymerase chain reaction: general methodology and recent advances.

In situ PCR is a new molecular technique, that combines the extreme sensitivity of PCR with the cellular localization provided by in situ hybridization (ISH), through the amplification of specific gene sequences within intact cells or tissue sections and increasing copy numbers to levels detectable by ISH or immunohistochemistry. In addition to the detection of viral DNA (CMV, HBV, HIV), we have used this technique for the study of DNA rearrangements, chromosomal translocations (t14;18) and viral RNA (HCV) in cells in suspension, cytocentrifuge preparations or archival tissue sections. We compared different approaches to in situ amplification of target sequences and visualization of PCR products by either subsequent ISH (indirect in situ PCR) or by direct detection of labeled nucleotides, which have been incorporated during PCR (direct in situ PCR). Our results indicate, that in situ PCR includes a number of different techniques, which are not equally applicable to all types of samples. In situ PCR appears to be most effective for the detection of DNA in single cell preparations with controlled fixation and pretreatment, although the quantification of results remains problematic. Artifacts caused by diffusion and extracellular generation of PCR products are a significant problem potentially leading to false positive results. In situ PCR works less efficiently in archival tissue sections due to poor quality of nucleic acids and retention of PCR products. Direct in situ PCR yields less specific results than indirect in situ PCR and requires additional controls such as omission of primers in the reaction mixture to detect artifacts.(ABSTRACT TRUNCATED AT 250 WORDS)

[Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies]. / Vergleich von Histologie, Immunhistochemie, RT-PCR, in situ Hybridisierung, und in situ RT-PCR zum Nachweis von Hepatitis C Virus in Paraffin-eingebettetem Lebergewebe.

To compare immunohistochemical and molecular methods for the detection of hepatitis C virus (HCV) infection in archival liver biopsies we analyzed formalin-fixed and paraffin-embedded liver specimens of 10 patients with serologically confirmed HCV infection. Methods employed included indirect FITC-immunofluorescence, reverse-transcriptase polymerase chain reaction (RT-PCR) using extracted RNA and Southern blotting with chemiluminescence-based detection, non-radioactive in situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes, direct in situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and indirect in situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. (Gastroenterology 1993; 104-595) together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies are representative; (2) the commercially available mAB TORDJI-22 appears to cross-react with non-HCV epitopes; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in situ RT-PCR or ISH; and (5) direct in situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. Further studies are required to define both optimal methods for sample processing and improvements of protocols, in order to increase detection sensitivity and specificity of HCV infection by immunohistochemical and molecular methods.

Expression of public idiotypes in patients with Lyme arthritis.

OBJECTIVE: Joints are often affected in Lyme disease and in some instances this may be due to immune autoreactivity. To characterise further the immune response in this disease investigations were carried out to determine the expression of three public idiotypes on serum immunoglobulins in patients with Lyme disease during the development of varying degrees of arthritis. METHODS: The expression of idiotypes (Ids) 16/6, BEG2, and PR4, first identified on monoclonal antibodies to DNA, was determined by an enzyme linked immunosorbent assay (ELISA) in serial blood samples from 12 patients with Lyme disease over a mean period of six years during the development of a variety of arthritic symptoms, and in serum samples from healthy control subjects and control subjects with systemic lupus erythematosus. RESULTS: Expression of serum IgM or IgG public Ids 16/6 and BEG2 was significantly increased in patients with Lyme disease. IgA Id 16/6 expression, in contrast, was significantly increased only during episodes of arthritis and was also related to its severity. IgM and IgG Id 16/6 expression was related to their respective total immunoglobulin concentration and, in the case of IgM, to the level of IgM antibodies to Borrelia burgdorferi, whereas similar findings were not apparent with IgA antibodies. This may indicate that the IgA response is related to the pathogenesis of arthritis, especially as total IgA and IgA Id 16/6 levels were found to increase over the duration of disease. Sequential analysis of antibodies also showed restriction in the expression of Id 16/6 as it was never found on all immunoglobulin isotypes at the same time, and Id PR4 was never expressed. Ids 16/6 and BEG2 expression, however, may be associated as seven patients expressed these idiotypes simultaneously. CONCLUSIONS: These data indicate the use of public idiotypes in the immune response against B burgdorferi, which may be restricted in terms of idiotype class and isotype expression, and a possible association between IgA antibodies bearing Id 16/6 with arthritis.

Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations.

Different approaches to the in-situ polymerase chain reaction (in-situ PCR) were compared in the detection and in-situ localization of chromosomal translocations (t14; 18) immunoglobulin gene rearrangements and viral DNA (cytomegalovirus, hepatitis B-virus) in cell suspensions, cytospins and tissue sections. Single and multiple primer pairs were compared in the amplification step of indirect in-situ PCR and long genomic probes or internal oligonucleotide probes in the subsequent in-situ hybridization (ISH). For direct in-situ PCR, in which amplification products were directly labeled with digoxigenin-11-dUTP during PCR and detected immunohistochemically, only single primer pairs were used for amplification. In-situ PCR yielded best results in the cell suspensions and worked less efficiently in cytospins or tissue sections. Quantification of the results obtained in artificial cell mixtures yielded only an approximate correlation between the number of expected and observed positive cells. The specificity of the results was greater with indirect in-situ PCR than direct in-situ PCR, where false positive results were frequent. Successful indirect in-situ PCR in tissue sections required the use of multiple primer pairs for amplification and genomic probes for detection by ISH. False positive results in direct in-situ PCR were caused by primer-independent, but DNA polymerase- and cycling-dependent incorporation of digoxigenin-labeled nucleotides into cellular DNA, possibly related to DNA repair and/or internal priming. Non-specific results were most marked in tissue sections and were much less frequent in cell suspensions. In-situ PCR includes a number of different techniques, which are not equally applicable to different starting materials. Accurate interpretation of the results requires vigorous controls.

In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique.

The in-situ polymerase chain reaction (in-situ PCR) is a novel molecular technique that combines the extreme sensitivity of the PCR with the anatomical localization provided by in-situ hybridization. A number of groups have recently reported studies using in-situ PCR for the detection of specifically amplified single-copy nucleic acid sequences in single cell preparations or low copy DNA sequences in tissue sections. In this overview, we describe the principles of in-situ PCR, review the applications of this technique and discuss future aspects of in-situ PCR. We critically compare the different in-situ PCR protocols described in the literature. Emphasis is placed on the absolute requirement for controls to allow accurate interpretation of results and the possible problems and pitfalls of the in-situ PCR methods, including artefacts related to diffusion of PCR products and non-specific incorporation of labelled nucleotides into fragmented DNA undergoing repair. It is concluded that this technique will eventually play an important role in specialized diagnostic laboratories in the evaluation of viral diseases, haematological and other malignancies which have unique genetic markers.

VH-gene representation in autoantibodies reflects the normal human B-cell repertoire.

The recurrence in the V-gene repertoire of individual germline VH genes can now be extended from the restricted B-cell populations of the fetus, autoantibodies and B-cell malignancies to the expressed V-gene repertoire of normal adults. Why the human B cell preferentially utilizes these individual VH genes remains speculative. However, it is apparent that the population of VH genes used to encode autoantibodies reflects the normal expressed repertoire (Fig. 7). Even so, the overrepresentation of other V genes such as Dxp'1 in anti-DNA antibodies and the presence of somatic mutation in the pathogenic autoantibodies of autoimmune disease continues to suggest an antigenic influence on V-gene selection. We postulate that only a fraction of available germline V genes are utilized in the expressed repertoire, and that polyspecificity of naturally occurring antibodies and somatic mutation of CDR3 compensate for the loss of diversity entailed by the limited use of the potential repertoire. The mechanisms by which germline genes become pathogenic remains unclear but they presumably relate to mutation, loss of regulatory control or perhaps environmental factors (Isenberg et al. 1992). What then are the mechanisms which lead to escape of these VH genes from normal control? What antigenic drive if any produces anti-DNA specificity in SLE? Why indeed is the expressed repertoire using only a fraction of the available germline? To answer these questions, further study of the V-gene repertoire of selected populations of antigen-binding cells and of pathogenic IgG autoantibodies is necessary and is ongoing. The contribution of individual V genes to antigen binding and idiotype is also being dissected and promises to yield important information about the relative contribution of VH genes to autoimmunity.

Relationship of antiphospholipid antibodies to pregnancy loss in patients with systemic lupus erythematosus: a cross-sectional study.

To determine whether an association exists between the presence of antiphospholipid antibodies and pregnancy loss, a cross-sectional study was performed. Consecutive women who were referred to three outpatient rheumatology clinics and who had systemic lupus erythematosus (SLE) and a history of one or more pregnancies were evaluated. Patients were interviewed to determine outcomes of all previous pregnancies. Blood was taken on two separate occasions at least 3 months apart to test for the presence of the lupus anticoagulant and anticardiolipin antibodies; on both occasions, five tests of the lupus anticoagulant, with well-defined normal ranges, and an enzyme-linked immunosorbent assay to measure IgG anticardiolipin antibodies were performed. Patients were considered to be positive for the lupus anticoagulant if one or more tests was abnormal on both occasions and positive for anticardiolipin antibodies if the test was abnormal on both occasions. Forty-two women were studied. Statistically significant associations were shown between lupus anticoagulant positivity and previous pregnancy loss (odds ratio [OR], 4.8; 95% confidence intervals [CI], 1.0 to 23.6; P = .05) and between anticardiolipin antibody positivity and previous pregnancy loss (OR, 20.0; 95% CI, 1.3 to 97.0; P = .01). All seven women with multiple episodes of pregnancy loss were lupus anticoagulant positive and four of these were also anticardiolipin antibody positive. If patients who are transiently positive for lupus anticoagulant and/or anticardiolipin antibodies are considered to be test positive, the associations with pregnancy loss are no longer statistically significant. Within the group of lupus anticoagulant-positive patients, we observed stronger associations between the presence of six or more positive tests and pregnancy loss than between the presence of two to five positive tests and pregnancy loss. No single test for the lupus anticoagulant provides a statistically significant association with pregnancy loss. The results of our study show that by performing multiple lupus anticoagulant tests and by repeating testing for lupus anticoagulant and anticardiolipin antibodies on more than one occasion, significant associations between the presence of antiphospholipid antibodies and previous pregnancy loss can be shown in patients with SLE.

In situ polymerase chain reaction detection of viral DNA, single-copy genes, and gene rearrangements in cell suspensions and cytospins.

The study of low-copy viral or genomic DNA sequences by in situ hybridization (ISH) is often limited by sensitivity. Using the polymerase chain reaction (PCR) for the amplification of target DNA sequences in fixed cells [in situ PCR] (ISPCR) before ISH, we have been able to greatly improve the sensitivity of ISH. Viral DNA present in low copy number, single-copy genes, as well as immunoglobulin gene rearrangements (VH3 family genes), were successfully amplified in cells in suspension or on glass slides (cytospins). Single primer pairs were used in the in situ amplification step and 35S- or digoxigenin-11-dUTP-labeled region specific oligonucleotide probes were used for detection of amplificants by ISH. Artifacts, presumably resulting from leakage of in situ amplificants out of cells, may be a significant problem in selected instances. By optimal fixation and permeabilization of cells, limiting PCR cycle number, amplification of long DNA sequences, and/or incorporation of biotinylated dNTPs to produce bulkier amplificants together with washing of cells after ISPCR, diffusion artifacts were significantly reduced. Probe hybridization to single-stranded long PCR fragments or messenger RNA were excluded as a source for false-positive ISPCR results. The techniques reported dramatically increase the sensitivity of ISH in the detection of low-copy viral infection as well as in the study of gene rearrangements, and provide unique opportunities to study chromosomal translocations and point mutations at the cellular level.

High-specificity in-situ hybridization. Methods and application.

We describe a technique of in-situ hybridization using oligonucleotide probes employing the expression of immunoglobulin VH genes as a model. Optimal conditions for hybridization with the 35S-labeled oligonucleotide probes were established with monoclonal B-cell lines that express VH genes of known nucleic acid sequence. The range of sensitivity and specificity achieved with this technique is documented. Under conditions of high stringency, this method can detect the expression of highly related VH hypervariable regions.

The relationship of antiphospholipid antibodies to thromboembolic disease in systemic lupus erythematosus: a cross-sectional study.

In order to determine whether an association exists between antiphospholipid antibodies (APLA) and thromboembolic events in patients with systemic lupus erythematosus (SLE), we performed a cross-sectional study of consecutive unselected SLE patients. The occurrence of previous thromboembolic events was determined by investigators blinded to the APLA status of the patients by critical review of objective tests that had been performed at the time of symptomatic presentation and by performing venous Doppler ultrasound of the legs to elicit venous reflux as an indication of previous venous thrombosis. The presence of APLA was determined by coagulation assays for the lupus anticoagulant (LA) using five tests with well-defined control ranges and by ELISA assay for anticardiolipin antibodies (ACLA). These tests were measured on two separate occasions. The results of the study demonstrate a statistically significant association between persistently abnormal ACLA assays and thromboembolic events and a non-significant trend between persistently abnormal LA and thromboembolic events. Transient abnormalities of LA and ACLA were less strongly associated with thromboembolic events. We conclude that in patients with SLE, there is a significant association between thromboembolism and APLA.