Subintimal Ki-67 as a synovial tissue biomarker for inflammatory arthropathies.

OBJECTIVES: Ki-67 is expressed in the nuclei of dividing cells and can be used to assess proliferation of synovial inflammatory and stromal cells. We evaluated subintimal Ki-67+ cell density as a tissue biomarker for inflammatory arthropathies and compared it to subintimal CD68, a synovial biomarker of RA. METHODS: Subintimal Ki-67+ and CD68+ cell densities were measured immunohistochemically in synovial specimens obtained from patients with rheumatoid arthritis (RA; n = 19), osteoarthritis (OA; n = 18), "non-inflammatory" orthopaedic arthropathies (avascular necrosis, meniscus injury, femur fracture; n = 16), chronic septic arthritis (n = 9), and histologically normal synovium (n = 10). RESULTS: were correlated with a histological synovitis score. Utilising the areas under receiver operating characteristic curves (AUCs), we compared the abilities of Ki-67 and CD68 to differentiate among these arthropathies. Results: Ki-67 was expressed widely in the subintimal of inflamed specimens and in RA pannus invading hard tissues. Compared to normal controls, it was highly overexpressed in RA (26.6-fold) and chronic septic arthritis (55-fold), and mildly elevated in OA (3.9-fold) and orthopaedic arthropathies (2.1-fold). Ki-67 and CD68 differentiated similarly well between RA and OA (AUC: Ki-67 = 0.91, CD68 = 0.94), Ki-67 better between chronic septic arthritis and RA, and CD68 better between OA and normal controls. Ki-67 (r = 0.80) and CD68 (r = 0.79) correlated positively with the synovitis score. CONCLUSIONS: Subintimal Ki-67 was overexpressed in inflammatory arthropathies, distinguished among differentially inflamed arthropathies, and correlated positively with the histological severity of synovitis. It may prove useful in synovial tissue classification and as a synovial marker of disease activity in clinical trials when biopsies are available.

The synovitis of "non-inflammatory" orthopaedic arthropathies: a quantitative histological and immunohistochemical analysis.

OBJECTIVE: To quantify inflammatory changes in synovial membranes from orthopaedic "non-inflammatory" arthropathies (Orth. A). METHODS: Synovial membranes from patients with femur fracture, avascular necrosis of the femur, plica syndrome, and meniscus and/or ligament injury (n = 23); rheumatoid arthritis (n = 28); osteoarthritis (OA; n = 25); and from normal controls (n = 10) were assessed by light microscopy, a histological synovitis score, immunostaining for CD3, CD20, CD38, CD68, Ki-67 and von Willebrand factor, and with an immunohistochemical inflammation score. RESULTS: Orth. A histology varied between normal and markedly inflamed. Predominant abnormalities were mild lining hyperplasia, scattered inflammatory cells and small perivascular infiltrates. The synovitis score classified Orth. A as "mild synovitis". Inflammatory cells occurred frequently: CD68+ cells in 100% of Orth. A specimens; CD3+, 91%; CD38+, 70%; and CD20+, 39%. Orth. A had 36% greater lining thickness (p = 0.04), 40% higher vascular density (p = 0.009) and 51.3-fold higher CD38+ cell density (p = 0.02) than normal controls; and 60% fewer subintimal Ki-67+ cells (p = 0.003), 42% fewer CD68+ lining cells (p<0.01) and 40% fewer subintimal CD68+ cells (p<0.01) than OA. The immunohistochemical inflammation score was 2.2-fold higher in Orth. A than in controls (p = 0.048) and similar to OA, with three Orth. A specimens showing marked inflammation. CONCLUSIONS: Synovial membranes from "non-inflammatory" arthropathies featured neovascularisation and inflammation intermediate between normal and OA synovium. These results expand previous findings that mechanical joint injury may lead to a mild-to-moderate synovitis.

A histomorphometric analysis of synovial biopsies from individuals with Gulf War Veterans' Illness and joint pain compared to normal and osteoarthritis synovium.

We compared histologic, immunohistochemical, and vascular findings in synovial biopsies from individuals with Gulf War Veterans Illness and joint pain (GWVI) to findings in normal and osteoarthritis (OA) synovium. The following parameters were assessed in synovial biopsies from ten individuals with GWVI: lining thickness, histologic synovitis score, and vascular density in hematoxylin & eosin-stained sections; and CD68+ lining surface cells and CD15+, CD3+, CD8+, CD20+, CD38+, CD68+, and Ki-67+ subintimal cells and von Willebrand Factor+ vessels immunohistochemically. Comparisons were made to synovial specimens from healthy volunteers (n = 10) and patients with OA or RA (n = 25 each). Histologic appearance and quantitative assessments were nearly identical in the GWVI and normal specimens. Vascular density was between 25% (H & E stains; p = 0.003) and 31% (vWF immunostains; p = 0.02) lower in GWVI and normal specimens than in OA. CD68+ macrophages were the most common inflammatory cells in GWVI (45.3 +/- 10.1 SEM cells/mm(2)) and normal synovium (45.6 +/- 7.4) followed by CD3+ T cells (GWVI, 15.1 +/- 6.3; normal, 27.1 +/- 9.2), whereas there were practically no CD20+, CD38+, and CD15+ cells. All parameters except lining thickness and CD15 and CD20 expression were significantly higher in OA. Five (20%) OA specimens contained significant fractions of humoral immune cells in mononuclear infiltrates, although the overall differences in the relative composition of the OA mononuclear infiltrates did not reach statistical significance compared to GWVI and normal synovium. In summary, the GWVI and normal synovia were indistinguishable from each other and contained similar low-grade inflammatory cell populations consisting almost entirely of macrophages and T cells.

A murine model of subglottic granulation.

OBJECTIVE: This study aimed to develop a functional model of subglottic stenosis by inducing direct airway irritation in transplanted mouse laryngotracheal complexes. METHODS: Laryngotracheal complexes from C57BL/6 mice were harvested and divided into three groups: uninjured, mechanically injured and chemically injured. Donor laryngotracheal complexes from each group were placed in dorsal subcutaneous pockets of recipient mice. Each week, the transplanted laryngotracheal complexes were harvested, and tissues were fixed, sectioned, and stained with haematoxylin and eosin. Representative slides were reviewed by a blinded pathologist, to determine the formation of granulation tissue, and graded as to the degree of granulation formation. RESULTS: Direct airway irritation induced granulation tissue formation under the disrupted epithelium of airway mucosa; this was seen as early as two weeks after chemical injury. CONCLUSION: Results indicate that granulation tissue formation in a murine model may be an efficient tool for investigating the development and treatment of subglottic stenosis.

Testicular cancer: an oncological success story.

Testicular cancer has become a model for a curable neoplasm. Our studies with cisplatin combination chemotherapy allow us to conclude that: (a) short-duration intensive induction therapy with the most active agents in optimal dosage is more important than maintenance therapy; (b) modest dose escalation increases toxicity without improving therapeutic efficacy; (c) it is possible to develop curative salvage therapy for refractory germ cell tumors; and (d) preclinical models predicting synergism, such as vinblastine + bleomycin or cisplatin + etoposide have clinical relevance. Finally, testicular cancer has also become a model for new drug development. Cisplatin was approved by the Food and Drug Administration for testis and ovarian cancer, and etoposide and ifosfamide were approved for refractory germ cell tumors. The success of these studies confirms the importance of the continued search for new investigational drugs in all solid tumors.

Outpatient varicocelectomy performed under local anesthesia.

AIM: To report a series of varicocelectomy performed under pure local anesthesia. METHODS: From July 1988 to June 2003, a total of 575 patients, aged between 15 and 73 years, underwent high ligation of the internal spermatic vein for treatment of a varicocele testis under a regional block in which a precise injection of 0.8 % lidocaine solution was delivered to involved tissues after exact anatomical references were made. A 100-mm visual analog scale (VAS) was used to assess whether the pain level was acceptable. RESULTS: The surgeries were bilateral in 52 cases, and unilateral in 523 cases. All were successfully performed on an outpatient basis except in the case of two patients, who were hospitalized because their surgeries required general anesthesia. Overall, 98.6 % (567/575) of men could go back to work by the end of the first post-operative week and only 8 (1.4 %) men reported feeling physical discomfort on the eighth day. The VAS scores varied from 11 mm to 41 mm with an average of (18.5+/-11.3) mm that was regarded as tolerable. CONCLUSION: This study has shown varicocelectomy under local anesthesia to be possible, simple, effective, reliable and reproducible, and a safe method with minimal complications. It offers the advantages of more privacy, lower morbidity, with no notable adverse effects resulting from anesthesia, and a more rapid return to regular physical activity with minor complications.

An improved method for oxidation of chromium(III) oxide-containing fecal samples by using sodium peroxide fusion.

A safer method of oxidation of Cr2O3-containing fecal samples from transit-time studies was developed using sodium peroxide to replace perchloric acid as the oxidizing agent. The percentage recovery of Cr2O3 with this method was compared with that of perchloric acid method for samples containing quantities of fecal ash and Cr2O3 typical of those from rodent transit-time studies. Both methods gave relatively constant percentage recoveries for Cr2O3 contents from 0.4 to 10 mg. Over this range, mean (+/- SD) percentage recoveries of Cr2O3 for sodium peroxide fusion and the perchloric acid method were 75.5 +/- 4.3 and 89.9 +/- 2.5, respectively. As long as percentage recovery is constant, the transit time as determined by calculation of the time of 80% excretion of the total recovered Cr2O3 is not affected. Sodium peroxide fusion provides a useful and safer alternative to perchloric acid oxidation in transit-time studies using Cr2O3 as a nonabsorbable marker.

Increased angiogenesis and cellular proliferation as hallmarks of the synovium in chronic septic arthritis.

OBJECTIVE: To characterize histologic alterations and inflammatory infiltrates in the synovium of patients with chronic septic arthritis (SeA). METHODS: Synovial membranes from patients with SeA (9 specimens; disease duration >4 weeks) were compared with specimens from patients with septic joint prosthesis loosening (septic total arthroplasty [SeTA]; 9 specimens), rheumatoid arthritis (RA; 25 specimens), osteoarthritis (25 specimens), and normal histology (10 specimens). Sections were stained with hematoxylin and eosin, tissue gram stain, and immunostains for von Willebrand factor (vWF; blood vessels), Ki-67 (dividing cells), CD15 (neutrophils), CD3 (T cells), CD20 (B cells), CD38 (plasma cells), and CD68 (macrophages). RESULTS: Gram stains were positive in all SeA and SeTA specimens. Mixed polymorphonuclear and mononuclear infiltrates predominated in SeA and SeTA. SeA could be differentiated from RA by higher densities of CD15+ cells (SeA:RA ratio 6.5:1; P < 0.001) or Ki-67+ cells (ratio 2.1:1; P = 0.012). The inflammatory infiltrate of SeTA was similar to SeA but contained fewer CD3+ cells (SeTA versus SeA 0.26:1; P = 0.009) and a tendency toward fewer CD20+ cells. Mean vascular density was strikingly increased in SeA (SeA:normal ratio 3.0:1; P < 0.001) and, to a lesser extent, in the vascularized areas of the SeTA specimens (SeTA:normal ratio 1.9:1). Ki-67/CD31 double immunostains demonstrated proliferating endothelial cells in small subintimal blood vessels, suggesting angiogenesis. Receiver operating characteristic curve analysis identified higher densities of CD15+ and Ki-67+ cells and vWF-positive vessels as histologic markers that differentiated SeA from RA. CONCLUSION: This first analysis of the synovium in patients with chronic pyogenic arthritis identified dramatic neovascularization and cell proliferation, accompanied by persistent bacterial colonization and heterogeneous inflammatory infiltrates rich in CD15+ neutrophils, as histopathologic hallmarks.

Absence of histologic evidence of synovitis in patients with Gulf War veterans' illness with joint pain.

OBJECTIVE: An unexplained multisymptom illness, Gulf War veterans' illness (GWVI), has been described among allied force veterans of the first Gulf War (1990-1991). It has been proposed that some of its symptoms reflect an immune dysfunction, and rheumatologic symptoms including joint pain and stiffness are reported frequently. However, it is unknown whether synovial inflammation causes the articular symptoms. We examined synovial tissue from individuals with GWVI and joint pain for evidence of inflammation. METHODS: We compared synovial biopsy samples from 6 individuals with GWVI and joint pain with samples from 9 clinically asymptomatic controls (hematoxylin and eosin [H&E] stains only) and biopsy samples or surgically obtained specimens from 10 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis (OA). Inflammatory changes were quantified in H&E stained sections with a modified synovitis score by immunostaining for CD3, CD20, CD38, CD68, Ki-67, and von Willebrand factor, and with a composite inflammation score based on these markers. RESULTS: Normal histology was seen in the GWVI specimens, except for mild focal lining hyperplasia and rare low-grade perivascular infiltrates in 1 specimen each. Mean +/- SEM synovitis scores were lowest and nearly identical in control (1.38 +/- 0.30) and GWVI specimens (1.41 +/- 0.29), intermediate in OA specimens (2.64 +/- 0.39), and highest in RA specimens (6.0 +/- 0.19). Likewise, inflammatory cells, cell division, vascular density, and composite inflammation score were lowest in the GWVI specimens. CONCLUSION: Despite significant joint pain, the GWVI synovia did not differ from normal controls. These results agree with other studies that have failed to document inflammatory or immunologic etiologies in GWVI.

The synergism of penile venous surgery and oral sildenafil in treating patients with erectile dysfunction.

A possible synergistic effect between penile venous surgery and oral sildenafil was inadvertently found in treating patients with erectile dysfunction in our clinic. We therefore sought to elucidate the possible synergic effect between venous surgery and sildenafil through studying patients who were non-responders preoperatively. From July 1998 to July 2003, 128 patients were diagnosed with veno-occlusive dysfunction. Subsequently, 65 of them underwent penile venous surgery and were assigned to the surgical treatment group. The remaining 63 men were assigned to the control group, and were subject to a simple re-exposure of oral sildenafil. All patients were evaluated with the international index of erectile function (IIEF-5) scoring. Sildenafil (12.5-100 mg) was prescribed postoperatively to all surgical patients as venous surgery alone was unsatisfactory and similarly, 100 mg preparation was prescribed for patients in the control group. The IIEF-5 scoring in the control group changed from a preoperative mean IIEF-5 score of 9.4 +/- 3.9 to 10.7 +/- 3.5 postoperatively. In surgical patients, however, the mean preoperative IIEF-5 score of 9.2 +/- 5.0, which increased to 15.1 +/- 5.0 (p < 0.001) postoperatively, further increased to 20.1 +/- 5.4 (p < 0.0001) after the addition of sildenafil. Although there was no significant difference between the two groups characteristics, there was a statistically significant difference between treatment results (p < 0.001). Overall, 61 men (93.8%) reported a positive response to sildenafil after venous stripping surgery. In contrast, only eight patients (12.7%) felt a beneficial response in the control group (p < 0.001). Forty-one of 65 patients had scores of > or =22, and 19 of these had a score of 25. No response was found in three (4.6%), and a decrease of 7 was seen in one (1.5%). In summary, patients in whom sildenafil was not effective preoperatively can become excellent responders after careful penile venous surgery. It appears that together, oral sildenafil and penile venous surgery may provide an encouraging solution to impotent patients with veno-occlusive dysfunction who are non-responders to sildenafil.

Butyrate increases colonocyte protein synthesis in ulcerative colitis.

Butyrate promotes epithelial cell healing and improves symptoms when administered rectally in patients with distal ulcerative colitis (UC). It was hypothesized that butyrate may enhance healing in patients with UC by stimulating colonocyte proliferation and/or protein production. Mucosa from the descending colon was obtained from patients with UC (n = 5), Crohn's disease (n = 8), diverticulitis (n = 6), and cancer (normal tissue 10 cm from tumor; n = 10). Epithelial cells were isolated using dispase/collagenase and differential sedimentation and incubated for 4 hr at 37 degrees C with either Na butyrate (10 mM) or NaCl (10 mM). Protein synthesis was assessed by [14C]leucine incorporation and proliferation was determined with [3H]thymidine. Mean viability and purity were >88%. Spontaneous proliferation was significantly increased in UC when compared to diverticulitis and normal controls. Butyrate significantly increased protein synthesis in UC epithelial cells when compared to saline control. The therapeutic effects of butyrate in patients with UC may be due to its use by epithelial cells as a metabolic fuel to increase protein production and promote healing.