A high tumour-stroma ratio (TSR) in colon tumours and its metastatic lymph nodes predicts poor cancer-free survival and chemo resistance

PurposeDespite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in tumour invasion and metastasis, the easy, low-cost and highly reproducible tumour-stroma ratio (TSR) could be a valuable prognostic marker, which is also believed to predict chemo resistance.MethodsTwo independent series of patients with colon cancer were selected. TSR was estimated by microscopic analysis of 4 µm haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low (≤ 50%) or TSR-high (> 50%). Differences in overall survival and cancer-free survival were analysed by Kaplan–Meier curves and cox-regression analyses. Analyses were conducted for TNM-stage I–II, TNM-stage III and patients with an indication for chemotherapy separately.ResultsWe found that high TSR was associated with poor cancer-free survival in TNM-stage I–II colon cancer in two independent series, independent of other known high-risk features. This association was also found in TNM-stage III tumours, with an additional prognostic value of TSR in lymph node metastasis to TSR in the primary tumour alone. In addition, high TSR was found to predict chemo resistance in patients receiving adjuvant chemotherapy after surgical resection of a TNM-stage II–III colon tumour.ConclusionIn colon cancer, the TSR of both primary tumour and lymph node metastasis adds significant prognostic value to current pathologic and clinical features used for the identification of patients at high risk of cancer recurrence, and also predicts chemo resistance.

A high tumour-stroma ratio (TSR) in colon tumours and its metastatic lymph nodes predicts poor cancer-free survival and chemo resistance.

PURPOSE: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in tumour invasion and metastasis, the easy, low-cost and highly reproducible tumour-stroma ratio (TSR) could be a valuable prognostic marker, which is also believed to predict chemo resistance. METHODS: Two independent series of patients with colon cancer were selected. TSR was estimated by microscopic analysis of 4 µm haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low (≤ 50%) or TSR-high (> 50%). Differences in overall survival and cancer-free survival were analysed by Kaplan-Meier curves and cox-regression analyses. Analyses were conducted for TNM-stage I-II, TNM-stage III and patients with an indication for chemotherapy separately. RESULTS: We found that high TSR was associated with poor cancer-free survival in TNM-stage I-II colon cancer in two independent series, independent of other known high-risk features. This association was also found in TNM-stage III tumours, with an additional prognostic value of TSR in lymph node metastasis to TSR in the primary tumour alone. In addition, high TSR was found to predict chemo resistance in patients receiving adjuvant chemotherapy after surgical resection of a TNM-stage II-III colon tumour. CONCLUSION: In colon cancer, the TSR of both primary tumour and lymph node metastasis adds significant prognostic value to current pathologic and clinical features used for the identification of patients at high risk of cancer recurrence, and also predicts chemo resistance.

Node negative colorectal cancer patients: assesment of high-risk features on recurrence.

BACKGROUND: The introduction of population-based screening programs for colorectal cancer (CRC) results in less patients with advanced disease. There is an increase in the amount of node negative CRC, which makes adequate risk stratification for this particular group of patients necessary. The addition of more risk factors to the conventional histological high-risk factors is investigated in this retrospective study. PATIENTS AND METHODS: A cohort of 227 node negative (stage I and II) CRC patients who were not treated with adjuvant chemotherapy were selected from two previously conducted cohort studies. Detailed histopathological examination was performed by two independent observers and molecular background (BRAF/RAS mutations, microsatellite status (MSI)) was studied. Univariate analyses were used to analyse differences in histological and mutational characteristics between patients with and without recurrence. P-values below 0.05 were considered statistically significant. RESULTS: Poorly differentiated histology (p:0.002), BRAF mutation (p:0.002) and MSI status (p:0.006) were found significant relevant risk factors that were related to recurrent disease. Poorly differentiated histology was associated with intermediate/high tumor budding (TB) (p:0.001), a BRAF mutation (p:0.001) and MSI status (p:0.001). A combination of all three features (poorly differentiated histology, BRAF and MSI) was more often present in the recurrence group. CONCLUSIONS: Recurrence in node negative CRC patients could be better predicted when molecular features such as, BRAF mutation and MSI status are incorporated into a model with poorly differentiated CRC. Therefore, these features might help in the selection of patients who possibly will benefit from adjuvant treatment.

Prognostic implications of occult nodal tumour cells in stage I and II colon cancer: The correlation between micrometastasis and disease recurrence.

INTRODUCTION: Occult nodal tumour cells should be categorised as micrometastasis (MMs) and isolated tumour cells (ITCs). A recent meta-analysis demonstrated that MMs, but not ITCs, are prognostic for disease recurrence in patients with stage I/II colon cancer. AIMS & METHODS: The objective of this retrospective multicenter study was to correlate MMs and ITCs to characteristics of the primary tumour, and to determine their prognostic value in patients with stage I/II colon cancer. RESULTS: One hundred ninety two patients were included in the study with a median follow up of 46 month (IQR 33-81 months). MMs were found in eight patients (4.2%), ITCs in 37 (19.3%) and occult tumour cells were absent in 147 patients (76.6%). Between these groups, tumour differentiation and venous or lymphatic invasion was equally distributed. Advanced stage (pT3/pT4) was found in 66.0% of patients without occult tumour cells (97/147), 72.9% of patients with ITCs (27/37), and 100% in patients with MMs (8/8), although this was a non-significant trend. Patients with MMs showed a significantly reduced 3 year-disease free survival compared to patients with ITCs or patients without occult tumour cells (75.0% versus 88.0% and 94.8%, respectively, p = 0.005). When adjusted for T-stage, MMs independently predicted recurrence of cancer (OR 7.6 95% CI 1.5-37.4, p = 0.012). CONCLUSION: In this study, the incidence of MMs and ITCs in patients with stage I/II colon cancer was 4.2% and 19.3%, respectively. MMs were associated with an reduced 3 year disease free survival rate, but ITCs were not.

Expression of CD105 on expanded mesenchymal stem cells does not predict their chondrogenic potential.

OBJECTIVE: Total bone marrow-derived mesenchymal stem cell (BMSC) populations differ in their potential to undergo chondrogenesis, with individual BMSCs differing in their chondrogenic capacity. The aim of this study was to explore the use of CD105 as a marker to isolate a chondrogenic subpopulation of BMSCs from the total, heterogeneous population. DESIGN: BMSCs were isolated from patients undergoing total hip replacement and following expansion (Passage 1-Passage 5), CD105 expression was investigated by FACS analysis. FACS was also used to sort BMSCs based on the presence of CD105 (CD105(+)/CD105(-)) or their amount of CD105 expression (CD105(Bright)/CD105(Dim)). After 3 or 5 weeks of differentiation, chondrogenic potential was determined by thionine staining for glycosaminoglycan (GAG) content and by detection of collagen type II using immunohistochemistry. RESULTS: Expanded total BMSC populations were composed almost exclusively of CD105(+) cells, the percentage of which did not correlate to subsequent chondrogenic potential; chondrogenic potential was observed to diminish with culture although CD105 expression remained stable. Similarly, differences in chondrogenic potential were observed between donors despite similar levels of CD105(+) BMSCs. Comparison of CD105(Bright) and CD105(Dim) BMSCs did not reveal a subpopulation with superior chondrogenic potential. CONCLUSIONS: Chondrogenic potential of BMSCs is often linked to CD105 expression. This study demonstrates that CD105 expression on culture expanded BMSC populations does not associate with a chondroprogenitor phenotype and CD105 should not be pursued as a marker to obtain a chondroprogenitor population from BMSCs.

The diagnostic value of one-step nucleic acid amplification (OSNA) for sentinel lymph nodes in colon cancer patients.

BACKGROUND: Lymph node status in colon cancer is critical for prognosis estimation and treatment allocation. The purpose of this study was to compare the performance of one-step nucleic acid amplification (OSNA) through detection of cytokeratin 19 mRNA levels with routine pathological examination (RP) and multilevel fine pathological examination (FP) in sentinel lymph nodes (SLN), detected using the ex vivo SLN mapping (SLNM) procedure, in presurgically defined nonmetastatic colon cancer patients. METHODS: In this prospective study, 325 SLNs of 128 patients from the Jeroen Bosch Hospital in 's-Hertogenbosch and the Leiden University Medical Center were investigated by RP (H&E), FP (H&E and Keratin Pan immunohistochemical staining), and OSNA. The SLNs were harvested by the SLNM procedure, using Patent blue or Indocyanine green. SLNs were divided and separate parts were used for RP, FP, and the OSNA assay. RESULTS: The diagnostic value of OSNA was 82.1 and 100 % for both FP and combined method (OSNA and FP) compared with RP. An upstaging rate of 20.2 % was obtained with the use of OSNA only and 36.4 % with the use of FP only. An upstaging rate of 46.5 % was obtained by combining the two methods together. CONCLUSIONS: OSNA and FP appeared to be promising tools for the detection of lymph node micro- and macrometastases in SLNs after SLNM. The performances of OSNA and FP in this study were superior to RP. Because OSNA allows analysis of the whole lymph node, sampling bias can be avoided. OSNA therefore may improve tumor staging.

Sentinel lymph node biopsy to direct treatment in gastric cancer. A systematic review of the literature.

Gastric cancer is one of the main causes of cancer-related deaths around the world. The prevalence of early gastric cancer (EGC) among all gastric cancers of 45-51% in Japan, but only 7-28% in Western countries. The prevalence of EGC is growing partly because of better diagnostics and screening programmes. Possible treatment options for EGC treatment are expanded by the introduction of endoscopic mucosal resection and endoscopic submucosal dissection Therefore, detailed knowledge about nodal metastatic risk is warranted. We performed a systematic review of the literature concerning studies investigating the role of sentinel lymph node biopsy in EGCr and whether there is enough proof to introduce SLN as a part of treatment for EGC in the Netherlands. Several detection substances (dye or radiocolloid) and injection methods (submucosal or subserosal) are investigated. An overall sensitivity percentage of 85.4% was found. In comparison, high and clinically sufficient percentages were observed for specificity (98.2%), negative predictive value (90.7%) and accuracy (94%). Subgroup analyses showed that the combination of dye and radiocolloid detection substances is the best method for sentinel lymph node detection in early gastric cancer. However, the precise method of sentinel lymph node biopsy in EGC has to be determined further. Large, randomized series should be initiated in Europe to address this issue.

Virus specific immune responses after human neoadjuvant adenovirus-mediated suicide gene therapy for prostate cancer.

PURPOSE: Neoadjuvant gene therapy potentially improves the outcome of primary treatment of prostate cancer by radical prostatectomy in patients with high risk of recurrence. We conducted a Phase I escalating dose study with a replication-defective adenovirus expressing the herpes simplex virus-thymidine kinase gene (Adv-HSV-tk vector). The primary end point was toxicity, while the evaluation of the patients' cellular and humoral immune responses served as a secondary endpoint. MATERIAL AND METHODS: The Adv-HSV-tk vector was injected into the prostate in two doses (2x10(10) to 2x10(11) viral particles), followed by ganciclovir twice daily for 14 days and retropubic radical prostatectomy on day 21. Adenovirus-specific neutralizing, IgG and IgA antibodies were evaluated. Peripheral blood mononuclear cells (PBMC) were stimulated by Adv-HSV-tk and analysed for IFN-gamma production and 3H-thymidine incorporation. Prostate specimens were immunostained for B (CD20+) and for T (CD3+) lymphocytes. RESULTS: Toxicity was minor in all 8 patients treated. In the prostate, no virus related cytopathic effect could be observed. Dose-dependent infiltration of T and B lymphocytes in the whole prostate and in tumor areas was observed. Boosting of adenovirus-specific antibody responses was observed in 7 patients, and an increased adenovirus-specific PBMC proliferation and IFN-gamma production was seen after Adv-HSV-tk stimulation. CONCLUSION: Neo-adjuvant adenovirus-mediated cytotoxic gene therapy prior to prostatectomy for prostate cancer is feasible and safe in an outpatient setting for intraprostatic vector doses up to 2x10(11) viral particles. Activation of the immune system was observed. Application of higher vector doses may be considered.

Short-term effects of whole-body vibration on maximal voluntary isometric knee extensor force and rate of force rise.

Whole-Body vibration (WBV) may lead to muscle contractions via reflex activation of the primary muscle spindle (Ia) fibres. WBV has been reported to increase muscle power in the short term by improved muscle activation. The present study set out to investigate the acute effects of a standard WBV training session on voluntary activation during maximal isometric force production (MVC) and maximal rate of force rise (MRFR) of the knee extensors. Twelve students underwent a single standard WBV training session: 5x1 min vibration (frequency 30 Hz, amplitude 8 mm) with 2 min rest in between. During vibration, subjects stood barefoot on the vibration platform with their knees at an angle of 110 degrees. At 90 s following vibration, maximal voluntary knee extensor force was reduced to 93 (5)% [mean (SD), P<0.05] of baseline value and recovered within the next 3 h. Voluntary activation remained significantly depressed (2-4%). Neither the electrically induced MRFR nor voluntary MRFR were significantly affected by WBV. In addition, six WBV training sessions in 2 weeks ( n=10) did not enhance either voluntary muscle activation during MVC [99 (2)% of the baseline value] or voluntary MRFR [98 (9)% of the baseline value]. It is concluded that in the short term, WBV training does not improve muscle activation during maximal isometric knee extensor force production and maximal rate of force rise in healthy untrained students.

Improved production and function of llama heavy chain antibody fragments by molecular evolution.

The aim of this study was to improve production level of llama heavy chain antibody fragments (V(HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V(HH) fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V(HH) with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V(HH) fragments were selected. We found that these shuffled V(HH) fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V(HH)s point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90 degrees C were compared with parental V(HH)s. One shuffled V(HH) was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V(HH) displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90 degrees C. Structural analysis suggested that improved antigen binding is associated with the A24 --> V24 substitution, which reduces the size of the hydrophobic pit at the llama V(HH) surface. We demonstrate that it is possible to improve desired characteristics of the same V(HH) fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.

Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama.

Functional heavy chain immunoglobulins have, so far, only been found in camels and llamas. Antigen-specific fragments of these heavy chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable and can be produced at high level by the yeast Saccharomyces cerevisiae. The work described in this paper was conducted to determine whether llamas (Lama glama) are a practical source of antigen-specific V(HH) fragments. Llamas were immunised with various types of antigens and the antibody responses were examined during the course of immunisation. Both, conventional and heavy chain IgG antibodies were produced in response to each of the antigens. The heavy chain IgG repertoire displayed a recognition pattern different to that of conventional llama IgGs, resulting in the expansion of the accessible epitope repertoire. Llamas have a lower proportion of heavy chain IgG antibodies in their serum than have camels. To enable the specific and efficient isolation of V(HH) genes from peripheral blood B-cells, the long and short-hinge sequences of Lama glama heavy chain IgGs were determined, revealing the presence of a novel subclass of short-hinge heavy chain IgG. Long and short-hinge specific PCR primers were designed to be used in the construction of llama V(HH) libraries. We conclude that, using the techniques described, antigen-specific V(HH) antibody fragments are readily accessible from the llama, thus providing highly valuable binding molecules for a variety of applications.

Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae.

Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.

Predictive value of sonographic examination to visualize retained placenta directly after birth at 16 to 28 weeks.

A prospective study was performed to assess the predictive value of an ultrasonographic examination directly after a spontaneous birth at 16 to 28 weeks' gestation to exclude the possibility of retained placental tissue. The aim of this procedure is to prevent routine curettage, which can induce Asherman's syndrome, uterine perforation, and anesthetic complications. Over a 2 year period the clinical course in 64 women, who had been delivered of their infants at 16 to 28 weeks' gestation, was followed through 6 weeks post partum. Sonographic examination was performed within 30 min after delivery of the placenta independent of macroscopic judgment of completeness of placenta. The examination was classified into three categories (with subsequent clinical interpretation): sharp lining of echogenic uterine wall with translucent cavity (uterine cavity containing fluid blood), sharp lining of the wall with echogenic area in cavity not continuous with the wall (uterine cavity with blood clot), and irregular lining with echogenic area continuous with the uterine wall and extending into the cavity (uterine cavity containing retained placental tissue). Women with sharp uterine lining without (n = 32) or with (n = 7) echogenicity in the cavity had no direct operative removal of placental tissue; 3 underwent curettage at a later stage (17, 18, and 34 days, respectively). A direct digital removal of placenta or curettage was performed on 25 women who revealed echogenicity continuous with the uterine wall. The 25 of 28 operatively obtained tissues were examined microscopically for trophoblasts. The sensitivity of the sonographic examination to find retained placental tissue was 85% (17 of 20) at 95% confidence intervals of 62 to 97%, the specificity was 88% (36 of 41) at 95% confidence intervals of 74 to 96%, and there were 25% (5 of 20) false positive judgments and 8% (3 of 39) false negative judgments. The positive predictive value of ultrasonography to find retained placenta of 68% (17 of 22) at 95% confidence interval of 55 to 92% combined with the negative predictive value of 92% (36 of 39) is sufficient to strongly suggest that curettage should not be performed routinely in these pregnancies at high risk for retained placental tissue.

Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies.

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.

Intracoronary-administered urapidil does not influence myocardial contractility, metabolic activity, or coronary sinus blood flow in humans.

OBJECTIVE: To compare the acute effect of intracoronary administration of urapidil and saline on myocardial contractility and metabolic activity. DESIGN: Prospective, controlled, open-label study. SETTING: University teaching hospital. PARTICIPANTS AND INTERVENTIONS: Eight patients with stable coronary artery disease (CAD) undergoing elective percutaneous transluminal coronary angioplasty (PTCA) received normal saline followed by urapidil, 4 mg, injected directly into the left main coronary artery. MEASUREMENTS AND MAIN RESULTS: Because local intracoronary administration is a non-steady-state condition, an in vitro model was used before the clinical experiments to establish the kinetic effects of acute administration of urapidil. The clinical experiments were performed in eight patients with CAD after PTCA. Measurements included a complete hemodynamic profile, coronary sinus blood flow (continuous thermodilution), left ventricular (LV) peak (+) dP/dt, LV peak (-) dP/dt, LV dP/dt/P(D)40, and LV end-diastolic pressures. Arterial and coronary venous blood samples were also obtained for the calculation of myocardial oxygen consumption. Baseline measurements I were first obtained, followed by intracoronary injection of 2 mL of saline. Additional measurements were obtained 1, 5, and 10 minutes after administration of saline. After a resting period (15 minutes), baseline measurements II, and intracoronary injection of urapidil, 4 mg (dissolved in 2 mL saline), additional measurements were obtained 1, 5, and 10 minutes later. Heart rate decreased 2.7+/-3.5 beats/min after injection of saline, whereas heart rate increased 2.0+/-1.8 beats/min after intracoronary urapidil, resulting in a significant difference in treatment effect (p = 0.003). There were no additional differences in treatment effect for any of the other measured or calculated parameters reflecting systemic hemodynamics, LV contractility, coronary dynamics, and myocardial metabolic activity. CONCLUSION: The results suggest that intracoronary bolus administration of preservative-free urapidil, 4 mg, is not associated with any detectable effect on myocardial contractility or coronary smooth muscle in awake nonsurgical patients with CAD, after PTCA.

White spot formation under orthodontic bands cemented with glass ionomer with or without Fluor Protector.

The purpose of this study was to determine whether an additional application of Fluor Protector before band cementation with glass ionomer cement reduces white spot formation compared with band cementation with glass ionomer cement. In the in vitro study, 80 premolars were divided in half, creating a control and a test group. All specimens were divided into four different groups to simulate different clinical situations and stored in a demineralizing solution to induce white spot formation. In the in vivo investigation, 18 orthodontic patients were incorporated in the study. One lower and one upper first molar band (randomly selected) were coated with Fluor Protector and then cemented with a glass ionomer cement (test group). The other two uncoated first molars were cemented with glass ionomer cement and served as the control group. The application of Fluor Protector in combination with Aquacem did not contribute to a reduction of white spot formation underneath molar bands compared with the use of Aquacem for banding.

Analysis of factors contributing to the formation of mononuclear cell aggregates ("escapees") in flow cytometric immunophenotyping.

During immunostaining, human lymphocytes may form aggregates with activated platelets and monocytes, resulting in increased forward (FSC) and sideward (SSC) light scatter signals. Consequently, aggregated cells "escape" from the standard FSC-SSC analysis gate, thereby producing erroneous results. We observed that the frequency of aggregate formation in peripheral blood mononuclear cell (PBMC) suspensions depended on cell donor, murine (m) monoclonal antibody (mAb) specificity and IgG subclass, type of fluorochrome conjugated to the mAb, amount of mAb used for immunostaining, time lapse between fixation of PBMC and flow cytometry, and other, as yet unidentified, factors. Platelets, monocytes, and granulocytes express the polymorphic class IIa IgG receptor (Fc gammaRIIa; CD32). A single amino acid difference, either arginine (R) or histidine (H) at amino acid position 131, underlies differential interaction with mIgG1. Because the Fc gammaRIIa-R131 allotypic form binds mIgG1 well in contrast to Fc gammaRIIa-H131, we studied the frequency of aggregate formation in PBMC suspensions from apparently healthy individuals allotyped for Fc gammaRIIa. The Fc gammaRIIa polymorphism contributed significantly to the frequency of mIgG1-induced cell aggregates, which was highest in Fc gammaRIIa-R/R131 individuals, intermediate in Fc gammaRIIa-R/H131 individuals, and lowest in Fc gammaRIIa-H/H131 individuals. The role of mIgG1-Fc gammaRIIa interactions in aggregate formation was confirmed by blocking Fc gammaRIIa by using F(ab')2 fragments of CD32 mAb. These data document the role of mIgG1 mAb binding by human class IIa IgG receptors in the formation of cell aggregates and show that inhibition of this interaction reduces this technical problem in flow cytometric immunophenotyping.

Steroid binding to human serum albumin and fragments thereof. Role of protein conformation and fatty acid content.

The binding properties of the steroids testosterone and pregnenolone to human serum albumin (HSA) and derived fragments of albumin have been investigated by means of equilibrium dialysis and circular dichroism. The 46 kDa peptic fragment (P46) of HSA comprises domains one and two of the HSA structure, whereas the other fragment, the 45 kDa tryptic fragment (T45) is composed of domains two and three. A comparison of the binding behaviour of the steroid ligands to HSA and its fragments showed that the single primary testosterone binding site in all probability is located in the second domain of the HSA molecule. For pregnenolone it was found that at least two primary binding sites are present, also located in domain two. Both steroids show pH dependent binding profiles in the case of HSA and the P46 fragment. The binding of the steroids to the T45 fragment seems to be pH independent. The same phenomenon was observed with the circular dichroism experiments, indicating a link between the steroid binding properties and the structural behaviour of the proteins. In fact, the binding properties of the steroids can be assigned to the neutral-to-base (N-B) transition. The possible role of fatty acids as modulators in the transport processes of steroids in the body is discussed.

Quantification of fluorescence properties of lymphocytes in peripheral blood mononuclear cell suspensions using a latent class model.

Lymphocytes, monocytes, granulocytes, and other blood cells can be distinguished on the basis of their forward (FSC) and sideward (SSC) light scatter properties and their expression of CD45 and CD14. A FSC,SSC gate can be set to include > 95% of the lymphocytes using a "back gating" procedure on the CD45+, CD14- cells. However, nonlymphoid cells such as monocytes have light scattering properties similar to lymphocytes. This problem occurs particularly in patient populations where the light scattering properties of lymphocyte subsets have changed (e.g., due to activation) and are similar to those of the monocytes. Thus, immunophenotyping using antibodies specific for other markers than CD45 and CD14 does not allow a direct assessment of the percentage of all lymphocytes positive for those markers. In order to optimize immunophenotyping we have developed analytic model in which the FSC,SSC dot plot is partitioned into six nonoverlapping light scatter regions. Each light scatter region contains a mixture population of different cell types, i.e., lymphocytes, monocytes, granulocytes, and other cells. The proportions of each cell type are known from the CD45,CD14 expression within each light scatter region. Under the assumption of independence of fluorescence and scatter properties conditional on cell type, the expression of markers other than CD45 or CD14 are derived from the cell type composition and the fluorescence properties on the other markers of each light scatter region. The underlying statistical model is a latent class model, and maximum likelihood estimates are computed using the expectation-maximization (EM) algorithm. The application of the model for immunophenotyping of lymphocytes of healthy individuals and cancer patients receiving immunotherapy is shown.

The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changes.

The effects of purified albumin species and albumin fragments (0.2-1% w/v) on short-term (4 h) steroid secretion by immature rat Leydig cells, in the presence of a maximally stimulating dose of luteinizing hormone (LH), were investigated. Human albumin and the peptic fragment (comprising residues 1-387) enhanced pregnenolone production in isolated rat Leydig cells, whereas chicken albumin and the tryptic fragment (comprising residues 198-585) were not active. This stimulatory effect of human albumin and the peptic fragment correlated with the potential of these proteins to undergo a pH-dependent neutral-to-base transition as measured by circular dichroism. The tryptic fragment and chicken albumin did not have the potential to undergo such a transition. The pH-dependent conformational changes of albumin and fragments thereof occurred in parallel with a change in the binding affinity for testosterone and pregnenolone. The fatty acid oleic acid and the drug suramin, only when present in a molar ligand-to-albumin ratio equal to or higher than 2, inhibited the albumin-mediated stimulation of steroid production. These data show that the stimulatory effects of albumin species on LH-induced Leydig cell pregnenolone production depend on their fatty acid content and correlate with the potential of these molecules to undergo conformational changes. It is unknown via which mechanisms albumin exerts its stimulatory effect, but the LH action through the cyclic AMP pathway seems not to be affected.