Integration of immunodeficiency virus in oocytes via intracytoplasmic injection: possible but extremely unlikely.

OBJECTIVE: To determine if human oocytes can be infected with HIV-1 via intracytoplasmic injection and to determine the infection threshold. DESIGN: Twenty-eight donated immature and unfertilized human oocytes from HIV-negative women were injected with 4 × 10(4) HIV-1 virions and 13 oocytes were used as uninjected controls. To determine the infection threshold, 543 cat oocytes were injected with 4 × 10(4), 4 × 10(2), or 40 copies of feline immunodeficiency virus (FIV) and 376 oocytes were used as controls. SETTING: Academic hospital. PATIENT(S)/ANIMAL(S): Donated immature human oocytes and mature cat oocytes. INTERVENTION(S): Injection with HIV-1 or FIV. MAIN OUTCOME MEASURE(S): Viral integration as measured by fluorescent in situ hybridization with HIV-1-specific probes or by nested FIV polymerase chain reaction. RESULT(S): We detected viral integration in three of 28 (11%) human oocytes injected with 4 × 10(4) copies of HIV-1. When injected with high dose FIV (4 × 10(4) copies) 16%-49% of cat oocytes showed viral integration. This decreased to 2%-7% and 0.6%-1.8% when an intermediate (4 × 10(2) copies) or low (40 copies) dose was injected, respectively. CONCLUSION(S): Human and cat oocytes can be infected with HIV-1 and FIV respectively, when injected with high amounts of virus. The probability of viral integration is extremely low when small amounts of virus particles are injected. Taking into account the small volume injected during intracytoplasmic injection, the chances of viral integration are 0.00002%.

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics.

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.

Activation of receptor for advanced glycation end products in osteoarthritis leads to increased stimulation of chondrocytes and synoviocytes.

OBJECTIVE: The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) could be one of these mechanisms. We undertook this study to investigate the role of the receptor for AGEs (RAGE) in mediating the cellular effects of AGEs on chondrocytes and fibroblast-like synoviocytes (FLS). METHODS: AGE levels in human cartilage were determined by fluorescence, browning, and pentosidine levels. Chondrocyte activation by AGEs was assessed as the release of proteoglycans and the synthesis of matrix metalloproteinase 1 (MMP-1) and type II collagen messenger RNA (mRNA). The activation of FLS by AGEs was measured by MMP-1 production and invasion through matrix proteins. RESULTS: Patients with focal degeneration of cartilage showed increased AGE levels in their healthy cartilage compared with the levels in healthy cartilage from donors without cartilage degeneration (P < 0.01 for both fluorescence and browning; P not significant for pentosidine content). Stimulation of bovine chondrocytes with glycated albumin increased the release of proteoglycans by 110% (P < 0.001) and the production of MMP-1 mRNA by 200% (P = 0.028). In addition, OA FLS produced 240% more MMP-1 when stimulated with glycated albumin (P < 0.001). Glycated matrix or albumin increased the catabolic activity of OA FLS, which was assessed as invasive behavior, by 150% and 140% (P = 0.001 and P = 0.010), respectively. Effects of stimulation with AGEs were blocked by a neutralizing antibody against RAGE, but not by an isotype control. CONCLUSION: This study shows that AGEs trigger RAGE on chondrocytes and FLS, leading to increased catabolic activity and therefore to cartilage degradation. AGEs, via RAGE, could therefore contribute to the development and/or progression of OA.