Germinal centers in human lymph nodes contain reactivated memory B cells.

To reveal migration trails of antigen-responsive B cells in lymphoid tissue, we analyzed immunoglobulin (Ig)M-V(H) and IgG-V(H) transcripts of germinal center (GC) samples microdissected from three reactive human lymph nodes. Single B cell clones were found in multiple GCs, one clone even in as many as 19 GCs. In several GCs, IgM and IgG variants of the same clonal origin were identified. The offspring of individual hypermutated IgG memory clones were traced in multiple GCs, indicating repeated engagement of memory B cells in GC reactions. These findings imply that recurring somatic hypermutation progressively drives the Ig repertoire of memory B cells to higher affinities and infer that transforming genetic hits in non-Ig genes during lymphomagenesis do not have to arise during a single GC passage, but can be collected during successive recall responses.

The pharmacokinetic behavior of the photosensitizer meso-tetra-hydroxyphenyl-chlorin in mice and men.

PURPOSE: Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is a hydrophobic photosensitizer that binds to plasma lipoproteins after intravenous injection. In vitro experiments with human plasma have shown that mTHPC initially binds to an unknown protein and subsequently redistributes to lipoprotein fractions. It has been suggested that this might explain the unusual pharmacokinetic profile of mTHPC humans. In humans, unlike in rodents, reappearance of mTHPC has been reported, resulting in a second plasma peak after intravenous injection. However, previous studies analyzed only limited time points during the first 24 h after injection. Our aim was to determine the pharmacokinetics of mTHPC in detail, and to investigate whether the pharmacokinetic behavior of the drug is affected by binding of mTHPC to lipoproteins in vivo. METHODS: Plasma of cancer patients and mice, intravenously injected with mTHPC, was analyzed for total drug content and drug distribution over the lipoprotein fractions. RESULTS: Pharmacokinetic profiles of mTHPC in a group of human subjects showed that apparent steady state drug levels were maintained for at least 10 h. Closer examination of individual profiles showed that the initial (5 min) plasma drug levels were on average 86% of the maximal plasma concentration, which occurred at about 5 h after injection. In mice, however, plasma pharmacokinetics were described by a standard bi-exponential decline of the drug concentration. The majority (>58%) of mTHPC injected into both BALB/c nude mice and patients initially bound to the HDL plasma fraction. We extended our study to ApoE -/- mice, with highly elevated lipoprotein levels, and SR-BI -/- mice, which are lacking the main clearance pathway for HDL associated cholesteryl esters, to take into account the differences between lipoprotein levels and clearance in mice and man. Although mTHPC distribution over the lipoproteins changed in these mice, pharmacokinetic profiles of mTHPC remained the same. CONCLUSIONS: We conclude that neither lipoprotein levels nor cholesterol metabolism affects the pharmacokinetics of mTHPC in plasma.

Photodynamic therapy in oncology.

Photodynamic therapy (PDT) is increasingly being recognized as an attractive, alternative treatment modality for superficial cancer. Treatment consists of two relatively simple procedures: the administration of a photosensitive drug and illumination of the tumor to activate the drug. Efficacy is high for small superficial tumors and, except for temporary skin photosensitization, there are no long-term side effects if appropriate protocols are followed. Healing occurs with little or no scarring and the procedure can be repeated without cumulative toxicity. Considering the efficacy and lack of long-term toxicity of PDT, and the fact that the first treatment of cancer with PDT was done more than 100 years ago, one might expect that this treatment had already become an established therapy. However, PDT is currently offered in only a few selected centers, although it is slowly gaining acceptance as an alternative to conventional cancer therapies. Here, we show the developmental steps PDT underwent and summarize the current clinical applications. The data show that, when properly used, PDT is an effective alternative treatment option in oncology.

Doppler optical coherence tomography to monitor the effect of photodynamic therapy on tissue morphology and perfusion.

We investigated the feasibility of using optical coherence tomography (OCT) for noninvasive real-time visualization of the vascular effects of photodynamic therapy (PDT) in normal and tumor tissue in mice. Perfusion control measurements were initially performed after administrating vaso-active drugs or clamping of the subcutaneous tumors. Subsequent measurements were made on tumor-bearing mice before and after PDT using the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). Tumors were illuminated using either a short drug light interval (D-L, 3 h), when mTHPC is primarily located in the tumor vasculature or a long D-L interval (48 h), when the drug is distributed throughout the whole tumor. OCT enabled visualization of the different layers of tumor, and overlying skin with a maximal penetration of < or =0.5-1 mm. PDT with a short D-L interval resulted in a significant decrease of perfusion in the tumor periphery, to 20% of pre-treatment values at 160 min, whereas perfusion in the skin initially increased by 10% (at 25 min) and subsequently decreased to 60% of pre-treatment values (at 200 min). PDT with a long D-L interval did not induce significant changes in perfusion. The concept of using noninvasive OCT measurements for monitoring early, treatment-related changes in morphology and perfusion may have applications in evaluating effects of anti-angiogenic or antivascular (cancer) therapy.

Optimizing meso-tetra-hydroxyphenyl-chlorin-mediated photodynamic therapy for basal cell carcinoma.

Meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown to be effective in the treatment of patients with multiple basal cell carcinoma (BCC). In the present study we further optimized the drug-light interval and examined the correlation between plasma drug levels and treatment efficacy. Thirteen patients with multiple BCC (a total of 366 lesions) were included in the study. Following intravenous administration of 0.1 mg kg(-1) mTHPC, lesions were illuminated with 10 J cm(-2) light (652 nm, 100 mW cm(-2)) at 12, 24, 48, 72 or 96 h. Plasma samples were taken prior to each illumination for determination of mTHPC levels, and tumor response was evaluated at 6 months and 1 year. Both univariable and multivariable analyses showed that optimal treatment outcome was obtained for a drug-light interval of 24 h when plasma drug levels were high. Overall, good cosmetic results with little or no scarring were obtained in 87% of the treated lesions and no serious side effects were observed. We optimized mTHPC-mediated PDT for patients suffering from multiple BCC by determining the most effective drug-light interval and showed that this treatment offers significant advantages over surgical resection.

Fluorescence detection of pleural malignancies using 5-aminolaevulinic acid.

STUDY OBJECTIVE: Although the use of video-assisted thoracoscopy has improved the diagnostic accuracy in patients presenting with pleural diseases, not all biopsies performed are conclusive and staging of the disease is not always optimal. Fluorescence diagnosis (FD) with 5-aminolaevulinic acid (5-ALA) has been used in the diagnostic workup for various malignancies. The impact of 5-ALA-mediated FD on diagnosis and staging during video-assisted thoracoscopy was examined. DESIGN: Prospective, single-center study. SETTING: National cancer center. PATIENTS: Twenty-six patients with nonconclusive pleural effusions who were scheduled for video-assisted thoracoscopy. INTERVENTION: Eligible patients were administered 1,500 to 2,500 mg po of 5-ALA before video-assisted thoracoscopy. After conventional inspection with white light, fluorescence inspection of the pleural cavity was performed (D-LIGHT Auto Fluorescent System; Karl Storz; Tuttlingen, Germany). Biopsy specimens of both normal and abnormal sites, as determined from white light and FD inspection, were obtained for histologic examination. RESULTS: One patient was ineligible, and two patients were not evaluable because of equipment failure. One postoperative death occurred due to preexisting myocardial disease. In another patient, an empyema developed; in another patient, a postoperative infection of the lung developed. Other toxicities were minimal. A definitive diagnosis was obtained in 24 of 25 cases, with malignant mesothelioma in 15 cases, other malignancies in 5 cases, one infection, and three benign diseases. Upstaging occurred in four patients (unsuspected tumor deposits) due to FD examination. In 23 patients, a total of 111 biopsy specimens could be analyzed. When correct findings of white light and FD were compared, FD had an additional value in 21 of 111 biopsies, compared to white light with 16 of 111 biopsies. CONCLUSIONS: FD using 5-ALA in the pleural cavity is feasible with limited side effects when used in addition to white light inspection. It improved visualization of abnormal lesions and led to upstaging in 4 of 15 mesothelioma patients.

Outcome of mTHPC mediated photodynamic therapy is primarily determined by the vascular response.

We have previously shown that the efficacy of photodynamic therapy (PDT) using the photosensitizer meso-tetra-hydroxyphenyl-chlorin (mTHPC) correlated with plasma drug levels at the time of illumination rather than drug levels in human tumor xenografts or mouse skin. These results suggested that vascular-mediated effects could be important determinants of PDT response in vivo. In the present study we further investigated the relationship between PDT response, mTHPC pharmacokinetics and the localization and extent of vascular damage induced in human squamous cell carcinoma xenografts (HNXOE). Plasma levels of mTHPC decreased exponentially with time after injection, whereas tumor drug levels remained maximal for at least 48 h. At 3 h after administration mTHPC was localized in the blood vessels, whereas at later times it was distributed throughout the whole tumor. Illumination at 3 h after mTHPC, which resulted in 100% long-term tumor cure, led to a marked reduction of vascular perfusion and increased tumor hypoxia at 1 h after treatment. Illumination at 48 h resulted in rapid regrowth of most tumors and only 10% cure. This protocol did not affect a significant decrease in vascular perfusion or increase in tumor hypoxia. These data show that optimal responses to mTHPC-mediated PDT were primarily dependent on the early vascular response, and that plasma drug levels at the time of illumination could predict this relationship.

Comparative sensitivity of microvascular endothelial cells, fibroblasts and tumor cells after in vitro photodynamic therapy with meso-tetra-hydroxyphenyl-chlorin.

The phototoxic effect of meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) on human microvascular endothelial cells (hMVEC) was compared with that on human fibroblasts (BCT-27) and two human tumor cell lines (HMESO-1 and HNXOE). To examine the relationship between intrinsic phototoxicity and intracellular mTHPC content, we expressed cell survival as a function of cellular fluorescence. On the basis of total cell fluorescence, HNXOE tumor cells were the most sensitive and BCT-27 fibroblasts the most resistant, but these differences disappeared after correcting for cell volume. Endothelial cells were not intrinsically more sensitive to mTHPC-PDT than tumor cells or fibroblasts. Uptake of mTHPC in hMVEC increased linearly to at least 48 h, whereas drug uptake in the other cell lines reached a maximum by 24 h. No difference in drug uptake was seen between the cell lines during the first 24 h, but by 48 h hMVEC had a 1.8- to 2.8-fold higher uptake than other cell lines. Endothelial cells showed a rapid apoptotic response after mTHPC-mediated PDT, whereas similar protocols gave a delayed apoptotic or necrotic like response in HNXOE. We conclude that endothelial cells are not intrinsically more sensitive than other cell types to mTHPC-mediated PDT but that continued drug uptake beyond 24 h may lead to higher intracellular drug levels and increased photosensitivity under certain conditions.