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Objectives: There is no consensus on the most reliable procedure to determine remission of cervical cancer after chemoradiotherapy (CRT). Therefore, this study aims to assess the diagnostic performance of two different imaging techniques, MRI and 18F[FDG]-PET/CT, in determining the presence of locoregional residual disease after CRT in patients with locally advanced cervical cancer., Methods: Patients diagnosed with locally advanced cervical cancer (FIGO 2009) treated with CRT were retrospectively identified from a regional cohort. The accuracy of MRI and 18F[FDG]-PET/CT in detecting locoregional residual disease was assessed with histology as the reference standard., Results: The negative predictive value (NPV) and positive predictive value (PPV) for locoregional residual disease detection of MRI and 18F[FDG]-PET/CT combined were 84.2% (95% CI 73.2-92.1), and 70.4% (95% CI 51.8-85.2), respectively. The NPV and PPV of MRI alone were 80.2% (95% CI 71.2-87.5) and 47.7% (95% CI 35.8-59.7), respectively, and values of 81.1% (95% CI 72.2-88.3) and 55.8 (95% CI 42.2-68.7), respectively, were obtained for 18F[FDG]-PET/CT alone., Conclusion: In this study, the reliability of MRI and 18F[FDG]-PET/CT in detecting locoregional residual disease was limited. Combining MRI and 18F[FDG]-PET/CT did not improve predictive values. Routine use of both MRI and 18F[FDG]-PET/CT in the follow-up after CRT should be avoided. MRI during follow-up is the advised imaging technique. Pathology confirmation of the presence of locoregional residual disease before performing salvage surgery is warranted.

Single-molecule localization microscopy requires sparse activation of emitters to circumvent the diffraction limit. In densely labeled or thick samples, overlap of emitter images is inevitable. Single-molecule localization of these samples results in a biased parameter estimate with a wrong model of the number of emitters. On the other hand, multiple emitter fitting suffers from point spread function degeneracy, which increases model and parameter uncertainty. To better estimate the model, parameters and uncertainties, a three-dimensional Bayesian multiple emitter fitting algorithm was constructed using Reversible Jump Markov Chain Monte Carlo. It reconstructs the posterior density of both the model and the parameters, namely the three-dimensional position and photon intensity, of overlapping emitters. The ability of the algorithm to separate two emitters at varying distance was evaluated using an astigmatic point spread function. We found that for astigmatic imaging, the posterior distribution of the emitter positions is multimodal when emitters are within two times the in-focus standard deviation of the point spread function. This multimodality describes the ambiguity in position that astigmatism introduces in localization microscopy. Biplane imaging was also tested, proving capable of separating emitters up to 0.75 times the in-focus standard deviation of the point spread function while staying free of multimodality. The posteriors seen in astigmatic and biplane imaging demonstrate how the algorithm can identify point spread function degeneracy and evaluate imaging techniques for three-dimensional multiple-emitter fitting performance., (© 2023. The Author(s).)

Three dimensional modulation-enhanced single-molecule localization techniques, such as ModLoc, offer advancements in axial localization precision across the entire field of view and axial capture range, by applying phase shifting to the illumination pattern. However, this improvement is limited by the pitch of the illumination pattern that can be used and requires registration between separate regions of the camera. To overcome these limitations, we present ZIMFLUX, a method that combines astigmatic point-spread-function (PSF) engineering with a structured illumination pattern in all three spatial dimensions. In order to achieve this we address challenges such as optical aberrations, refractive index mismatch, supercritical angle fluorescence (SAF), and imaging at varying depths within a sample, by implementing a vectorial PSF model. In scenarios involving refractive index mismatch between the sample and immersion medium, the astigmatic PSF loses its ellipticity at greater imaging depths, leading to a deterioration in axial localization precision. In contrast, our simulations demonstrate that ZIMFLUX maintains high axial localization precision even when imaging deeper into the sample. Experimental results show unbiased localization of 3D 80 nm DNA-origami nanostructures in SAF conditions with a 1.5-fold improvement in axial localization precision when comparing ZIMFLUX to conventional SMLM methods that rely solely on astigmatic PSF engineering.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\sigma$ and the RNAP $\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Modulation enhanced single-molecule localization microscopy (meSMLM) methods improve the localization precision by using patterned illumination to encode additional position information. Iterative meSMLM (imeSMLM) methods iteratively generate prior information on emitter positions, used to locally improve the localization precision during subsequent iterations. The Cramér-Rao lower bound cannot incorporate prior information to bound the best achievable localization precision because it requires estimators to be unbiased. By treating estimands as random variables with a known prior distribution, the Van Trees inequality (VTI) can be used to bound the best possible localization precision of imeSMLM methods. An imeSMLM method is considered, where the positions of in-plane standing-wave illumination patterns are controlled over the course of multiple iterations. Using the VTI, we analytically approximate a lower bound on the maximum localization precision of imeSMLM methods that make use of standing-wave illumination patterns. In addition, we evaluate the maximally achievable localization precision for different illumination pattern placement strategies using Monte Carlo simulations. We show that in the absence of background and under perfect modulation, the information content of signal photons increases exponentially as a function of the iteration count. However, the information increase is no longer exponential as a function of the iteration count under non-zero background, imperfect modulation, or limited mechanical resolution of the illumination positioning system. As a result, imeSMLM with two iterations reaches at most a fivefold improvement over SMLM at 8 expected background photons per pixel and 95% modulation contrast. Moreover, the information increase from imeSMLM is balanced by a reduced signal photon rate. Therefore, SMLM outperforms imeSMLM when considering an equal measurement time and illumination power per iteration. Finally, the VTI is an excellent tool for the assessment of the performance of illumination control and is therefore the method of choice for optimal design and control of imeSMLM methods., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μ m thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Single-photon avalanche diode (SPAD) arrays can be used for single-molecule localization microscopy (SMLM) because of their high frame rate and lack of readout noise. SPAD arrays have a binary frame output, which means photon arrivals should be described as a binomial process rather than a Poissonian process. Consequentially, the theoretical minimum uncertainty of the localizations is not accurately predicted by the Poissonian Cramér-Rao lower bound (CRLB). Here, we derive a binomial CRLB and benchmark it using simulated and experimental data. We show that if the expected photon count is larger than one for all pixels within one standard deviation of a Gaussian point spread function, the binomial CRLB gives a 46% higher theoretical uncertainty than the Poissonian CRLB. For typical SMLM photon fluxes, where no saturation occurs, the binomial CRLB predicts the same uncertainty as the Poissonian CRLB. Therefore, the binomial CRLB can be used to predict and benchmark localization uncertainty for SMLM with SPAD arrays for all practical emitter intensities.

Localization microscopy offers resolutions down to a single nanometer but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from the drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal precision and bias, which leaves room for improvement. Here, we minimize a bound on the entropy of the obtained localizations to efficiently compute a precise drift estimate. Within practical compute-time constraints, simulations show a 5x improvement in drift estimation precision over the widely used RCC algorithm. The algorithm operates directly on fluorophore localizations and is tested on simulated and experimental datasets in 2D and 3D. An open source implementation is provided, implemented in Python and C++, and can utilize a GPU if available.

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.

Background and Purpose: Small cell carcinoma of the esophagus (SCEC) is a rare subtype of esophageal cancer for which optimal treatment is unknown. We analyzed the impact of treatment factors on outcome in patients with nonmetastasized SCEC., Methods: Patients with a histologically confirmed SCEC without distant metastases were analyzed in a nationwide multicenter retrospective cohort. All patients received radiotherapy as part of curative treatment between January 2000 and December 2014. Details on treatment and outcome were retrieved from individual charts. Cox regression analysis was used to determine prognostic factors for survival., Results: Fifty-eight patients were analyzed. Median survival was 16 months (95% confidence interval, 11-21 mo). Infield recurrences occurred in 25%, distant metastases in 45%, and brain metastases in 12%. In total, 63% of patients developed a recurrence. Most recurrences (67%) occurred within 1 year. In univariable analyses an increased number of chemotherapy cycles (>3) and lower radiotherapy doses (<45 Gy) were associated with improved survival. T-stage, N-stage, treatment period, type of chemotherapy, prophylactic cranial irradiation, and age were not associated with survival. In multivariable analyses, only the number of chemotherapy cycles was associated with better survival (hazard ratio, 0.78; P=0.006)., Conclusions: SCEC recurs frequently at distant sites after definitive chemoradiotherapy and usually within 1 year after curative treatment. With a dose of 45 to 50 Gy, infield recurrence rate was low. We found a relationship between number of received chemotherapy cycles and survival with best results obtained after at least 4 cycles of chemotherapy.

Purpose: The purpose of this study was to analyze the effect of 2D conventional brachytherapy (CBT) compared to 3D MRI-guided brachytherapy (IGBT) with and without the use of interstitial needles on local control, overall survival, and toxicity in patients treated for cervical cancer with radiation or chemoradiation., Material and Methods: A retrospective analysis was performed of biopsy-proven FIGO IB-IVA cervical cancer patients, treated with primary radiation or chemoradiation, followed by brachytherapy (BT) between January 1997 and July 2016. Endpoints were local control, overall survival, and toxicity., Results: Of 126 patients included, 35 have been treated with CBT, 31 with IGBT without needles (IC), and 60 with IGBT with needles (ICIS). External beam radiotherapy (EBRT) had mostly been delivered concurrently with chemotherapy (weekly cisplatin). Overall local control was 93% after 1 year, and 88% after 3 years. Overall 3-year survival was 75%, and 5-year survival was 66%. The 3D technique (IGBT cohorts) showed a trend for an improved local control and overall survival ( p = 0.05) compared to the 2D technique (CBT cohort). A decrease in toxicity was observed from 17% (2D cohort) to 12% (3D cohort). The use of interstitial needles was associated with a higher high-risk clinical target volume (HR-CTV) dose (11.3 Gy vs. 9.9 Gy) and a lower D 2cc bladder dose (10.9 Gy vs. 14.7 Gy, both p < 0.01)., Conclusions: In cervical cancer treatment, the use of a 3D brachytherapy technique (MRI-guided with or without interstitial needles) showed a trend towards an increased local control and improved overall survival with reduced toxicity, compared to the conventional 2D brachytherapy technique. The use of interstitial needles allowed dose sculpting, resulting in delivery of higher doses to the HR-CTV, while reducing radiation doses to organs at risk, such as the bladder., Competing Interests: Authors report no conflict of interest.

The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To study the behavior of biological macromolecules and enzymatic reactions under force, advances in single-molecule force spectroscopy have proven instrumental. Magnetic tweezers form one of the most powerful of these techniques, due to their overall simplicity, non-invasive character, potential for high throughput measurements, and large force range. Drawbacks of magnetic tweezers, however, are that accurate determination of the applied forces can be challenging for short biomolecules at high forces and very time-consuming for long tethers at low forces below ∼1 piconewton. Here, we address these drawbacks by presenting a calibration standard for magnetic tweezers consisting of measured forces for four magnet configurations. Each such configuration is calibrated for two commonly employed commercially available magnetic microspheres. We calculate forces in both time and spectral domains by analyzing bead fluctuations. The resulting calibration curves, validated through the use of different algorithms that yield close agreement in their determination of the applied forces, span a range from 100 piconewtons down to tens of femtonewtons. These generalized force calibrations will serve as a convenient resource for magnetic tweezers users and diminish variations between different experimental configurations or laboratories.

USCOM is an ultrasound-based method which has been accepted for noninvasive hemodynamic monitoring in various clinical conditions (USCOM, Ultrasonic cardiac output monitoring). The present study aimed at comparing the accuracy of the USCOM device with that of the thermodilution technique in patients with septicemia. We conducted a prospective observational study in a medical but noncardiological ICU of a university hospital. Septic adult patients (median age 55 years, median SAPS-II-Score 43 points) on mechanical ventilation and catecholamine support were monitored with USCOM and PiCCO (n = 70). Seventy paired left-sided CO measurements (transaortic access = CO(US-A)) were obtained. The mean CO(US-A) were 6.55 l/min (±2.19) versus CO(PiCCO) 6.5 l/min (±2.18). The correlation coefficient was r = 0.89. Comparison by Bland-Altman analysis revealed a bias of -0.36 l/min (±0.99 l/min) leading to a mean percentage error of 29%. USCOM is a feasible and rapid method to evaluate CO in septic patients. USCOM does reliably represent CO values as compared to the reference technique based on thermodilution (PiCCO). It seems to be appropriate in situations where CO measurements are most pertinent to patient management.

We discuss the case of a 32 year-old male with severe microcytic anemia (hemoglobin 2,9 g/dl) and megaloblastic changes in the bone marrow. The patient reported of substantial dietary weight loss. The family history was positive for beta-thalassemia. Previous blood work showed iron deficiency with mild anemia. Further work-up verified beta-thalassemia minor and revealed severely decreased vitamin B12 levels with positive anti intrinsic-factor antibodies, pathognomonic for autoimmune pernicious anemia. The paradoxon therefore dissolved as a pernicious anemia with megaloblastic changes with microcytic erythrocytes due to beta-thalassemia.

Objective: To perform a systematic review of the literature on the accuracy of prediction models in the preoperative assessment of adnexal masses., Data Sources: Studies were identified through the MEDLINE and EMBASE databases from inception to March 2008. The MEDLINE search was performed using the keywords ["ovarian neoplasms"[MeSH] NOT "therapeutics"[MeSH] AND "model"] and ["ovarian neoplasms"[MeSH] NOT "therapeutics"[MeSH] AND "prediction"]. The Embase search was performed using the keywords [ovary tumor AND prediction], [ovary tumor AND Mathematical model], and [ovary tumor AND statistical model]., Methods of Study Selection: The search detected 1,161 publications; from the cross-references, another 116 studies were identified. Language restrictions were not applied. Eligible studies contained data on the accuracy of models predicting the risk of malignancy in ovarian masses. Models were required to combine at least two parameters., Tabulation, Integration, and Results: Two independent reviewers selected studies and extracted study characteristics, study quality, and test accuracy. There were 109 accuracy studies that met the selection criteria. Accuracy data were used to form two-by-two contingency tables of the results of the risk score compared with definitive histology. We used bivariate meta-analysis to estimate pooled sensitivities and specificities and to fit summary receiver operating characteristic curves.Studies included in our analysis reported on 83 different prediction models. The model developed by Sassone was the most evaluated prediction model. All models has acceptable sensitivity and specificity. However, the Risk of Malignancy Index I and the Risk of Malignancy Index II, which use the product of the serum CA 125 level, an ultrasound scan result, and the menopausal state, were the best predictors. When 200 was used as the cutoff level, the pooled estimate for sensitivity was 78% for a specificity of 87%., Conclusion: Based on our review, the Risk of Malignancy Index should be the prediction model of choice in the preoperative assessment of the adnexal mass.

Objective: To systematically review the medical literature reporting on ultrasound factors that can be predictive for the outcome of an attempt at external cephalic version (ECV)., Methods: MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials were searched. Studies reporting on potential ultrasound prognosticators and ECV success rates that allowed construction of a 2x2 table were selected., Results: We selected 37 primary articles reporting on 7709 women. Posterior placental location (odds ratio (OR), 1.9; 95% CI, 1.5-2.4), complete breech position (OR, 2.3; 95% CI, 1.9-2.8) and an amniotic fluid index>10 (OR, 1.8; 95% CI, 1.5-2.1) were predictors of successful ECV., Conclusion: Success of an ECV attempt is associated with ultrasound parameters such as fetal position, amniotic fluid and placental location. This knowledge can be used to develop a prognostic model to predict successful ECV., (Copyright (c) 2008 ISUOG.)

Objective: The objective of the study was to systematically review the medical literature reporting on potential clinical prognosticators for the outcome of external cephalic version (ECV)., Study Design: Medline, EMBASE, and Cochrane Central Register of Controlled Trials were searched. Studies reporting on potential clinical prognosticators and ECV success rates that allowed construction of a 2 x 2 table were selected., Results: We detected 53 primary articles reporting on 10,149 women. Multiparity (P >/= 1.00; odds ratio [OR], 2.5; 95% confidence interval [CI], 2.3-2.8), nonengagement of the breech (OR, 9.4; 95% CI, 6.3-14), a relaxed uterus (OR, 18; 95% CI, 12-29), a palpable fetal head (OR, 6.3; 95% CI, 4.3-9.2), and maternal weight less than 65 kg (OR, 1.8; 95% CI, 1.2-2.6) were predictors for successful external cephalic version., Conclusion: Success of an ECV attempt is associated with clinical factors. This should be taken into account in the counseling of women prior to an ECV attempt.

Objectives: To describe symptoms, delay in presentation and reasons for non-presentation among women diagnosed with benign, low malignant potential and malignant ovarian tumors., Methods: Study participants included 457 women who underwent surgery for an ovarian tumor in Queensland, Australia, between July 1999 and February 2002 (244 with invasive cancer, 62 with low malignant potential tumors, and 151 with benign ovarian tumors). Women were contacted a minimum of three months post-diagnosis. Information concerning symptoms and presentation history was obtained via interview., Results: Overall, only 8% of the women were asymptomatic at the time of their diagnosis. Women with invasive cancer reported a greater number of symptoms (3.1 and 3.6 for Stages I-II and III-IV, respectively) than women with benign or low malignant potential tumors (2.8 and 2.2 respectively; p < 0.0001). Women with invasive disease were more likely to experience weight loss or gain, general malaise, chest/respiratory pain, abdominal swelling and bowel symptoms than women with benign ovarian tumors, however the symptom pattern for early- and late-stage invasive ovarian cancer could not be clearly differentiated. There was no suggestion that women with advanced stage disease had delayed longer before presenting to their doctor. The most common reasons given for not telling their doctor about specific symptoms were the woman's perception that the symptom was not serious enough, it was mild or intermittent, or was related to normal physical changes associated with age or menopause., Conclusions: We found only marginal differences in the symptom patterns of early and advanced stage invasive cancer. Delay in presentation was not associated with more advanced disease suggesting that earlier diagnosis may not increase the proportion of cancers diagnosed at an early stage.

Patients with rheumatoid arthritis have decreased numbers of T mu lymphocytes in their peripheral blood. To find out whether these low number of T mu lymphocytes were associated with the presence of anti-lymphocyte antibodies, the sera of 27 patients with definite or classical rheumatoid arthritis (RA) were investigated for the presence of autoantibodies against subsets of lymphocytes. In addition the numbers of T, T mu, T gamma and B lymphocytes in the peripheral blood of these patients were investigated. Patients with active RA showed lower numbers of T mu lymphocytes in their peripheral blood than patients with inactive RA. However, both groups of RA patients had significantly decreased numbers of T mu lymphocytes in their peripheral blood as compared with 22 age matched healthy donors. Moreover, mainly in patients with active RA cold reactive antibodies were found directed against T mu and B lymphocytes, but never against T gamma lymphocytes of healthy donors. Similar results were found in the indirect immunofluorescence procedure when tested for reactivity against T-cell subsets. This serum reactivity was not caused by rheumatoid factors or antinuclear antibodies. Since RA sera after precipitation with 2.5% polyethyleneglycol, still showed cytotoxicity against T and B lymphocytes, it is suggested that this serum reactivity is not caused by immune complexes but by antibodies.

A mixed rosette assay is described for simultaneous detection of T lymphocytes and lymphocytes bearing receptors for the Fc Fragments of IgM (RFc mu) or IgG (RFc gamma) in unfractionated lymphocyte suspensions. For optimal detection of T lymphocytes, treatment of sheep erythrocytes with neuraminidase was necessary. ox erythrocytes labelled with fluorescein isothiocyanate and sensitized with either rabbit IgM or rabbit IgG anti-ox antibodies served as indicator erythrocytes for RFc mu or RFc gamma bearing lymphocytes. In periphral blood the percentage of RFc mu bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37 degrees C. Ratios of non-fluoresceinated sheep erythrocytes to senanti-ox antibodies served as indicator erythrocytes for RFc mu or RFc gamma bearing lymphocytes. In periphral blood the percentage of RFc mu bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37 degrees C. Ratios of non-fluoresceinated sheep erythrocytes to senanti-ox antibodies served as indicator erythrocytes for RFc mu or RFc gamma bearing lymphocytes. In periphral blood the percentage of RFc mu bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37 degrees C. Ratios of non-fluoresceinated sheep erythrocytes to sensitized fluoresceinated ox erythrocytes are critical in the mixed rosette assay; for simultaneous detection of T lymphocytes and RFc mu bearing lymphocytes 1 : 1, and for T lymphocytes and RFc gamma bearing lymphocytes 2 : 1. In both assays the rosette suspension was preincubated for 10 min at 37 degrees C and then centrifuged at 200 X g for 5 min. For optimal simultaneous detection of T lymphocytes and RFc mu bearing lymphocytes incubation at room temperature for at least 2 h was necessary and for T lymphocytes and RFc gamma bearing lymphocytes incubation at room temperature for at least 2 h was necessary and for T lymphocytes and RFc gamma bearing lymphocytes incubation at 4 degrees C for at least 2 h was essential. The percentages of RFc mu or RFc gamma bearing lymphocytes (T mu and T gamma respectively) in the T lymphocyte population determined by mixed rosette assay did not differ significantly from the percentages of RFc mu and RFc gamma bearing lymphocytes in lymphocyte suspensions enriched in T cells by E-rosette sedimentation. The mixed rosette assay is suitable for detection of T mu and T gamma cells in immune deficiency and auto-immune diseases and for analysis of the mononuclear cells in lymphoreticular malignancies.

Cerebriform mononuclear cells (CMC) constitute a morphologically distinct subpopulation of T cells in healthy individuals. They are characterized ultrastructurally by a highly indented nucleus, a high nucleus-to-cytoplasm ratio, condensed chromatin along the nuclear membrane and a scanty cytoplasm. In order to characterize the peripheral blood CMC by enzyme-histochemistry and membrane characteristics, lymphocyte fractions enriched for T cells, T mu cells, T gamma cells and T cells without Fc mu and Fc gamma receptors (T0 cell fraction), or depleted of T cells, were investigated for the presence of alpha-naphthylacetate esterase and acid phosphatase at light and electron microscopic levels. CMC were found exclusively in the T mu-enriched and T0 cell fraction, indicating that these cells have either an Fc mu receptor or no Fc receptor at all. Except for their nuclear indentation, both the CMC in the T mu-enriched fractions and the CMC in the T0 fractions ultrastructurally resembled the characteristic cell in the T mu fraction (TM-type cell), but differed from the characteristic cell in the T gamma fraction (TG-type cell). Moreover, like the TM-type cells all CMC showed paranuclear dots of alpha-naphthylacetate esterase and acid phosphatase activity in their cytoplasm. From these observations it was concluded that CMC without Fc mu receptors were either stimulated T mu cells or precursor T mu cells. Thus CMC in healthy individuals constitute a distinct subpopulation of T cells not only morphologically but also histochemically and immunologically.

Peripheral blood lymphocytes (PBL) and synovial fluid lymphocytes ( SFL ) of patients with rheumatoid arthritis (RA) were examined with monoclonal antibodies, with coated ox red blood cells for the expression of Fc receptors for IgG or IgM (T mu and T gamma cells), and incubated for the demonstration of alpha- naphtyl acetate esterase and acid phosphatase. Equal percentages of OKT4 and OKT8 PBL were found in clinically active and inactive RA patients, and in healthy controls, but decreased percentages of OKT4 and increased percentages of OKT8-positive lymphocytes were found among the SFL . The percentages of T mu and T gamma cells, the presence of HLA-DR membrane antigens on T lymphocytes as well as the staining pattern for the enzymes revealed that SFL of patients with RA were highly activated, compared to PBL of RA patients and healthy controls. It can be concluded from this study that a single determination of OKT4 and OKT8-positive lymphocytes in the peripheral blood of RA patients has no predictive value for disease activity. However, the results of the experiments on T lymphocyte-activation clearly showed preferential activation of SFL compared to PBL, indicating that activation of lymphocytes occurs at the site of inflammation.

The number of various lymphocyte subpopulations in the peripheral blood of 28 patients with definite rheumatoid arthritis (RA) were studied. The results were correlated with the disease activity, as assessed by the Ritchie index, erythrocyte sedimentation rate and clinical impression. Patients with active RA showed decreased numbers of T mu lymphocytes, strongly increased numbers of Tnull lymphocytes and slightly increased percentages of Fc gamma lymphocytes as compared to 22 healthy donors. Patients with inactive RA had similar, less striking, but significant changes in T mu and Tnull lymphocytes, but in contrast to patients with active RA had Increased numbers of T gamma lymphocytes. The imbalances in T mu and Tnull cells in patients with RA might be explained by endogenous T mu cell activation, resulting in increased numbers of Tnull cells.