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Brendon J Coventry1, Michael J Weightman1, John M Skinner2, John Bradley31Discipline of Surgery, University of Adelaide; 2Department of Pathology; 3Department of Clinical Immunology, Flinders University, Adelaide, SA, AustraliaAbstract: Quantitation of cell density in tissues has proven problematic over the years. The manual microscopic methodology, where an investigator visually samples multiple areas within slides of tissue sections, has long remained the basic 'standard' for many studies and for routine histopathologic reporting. Nevertheless, novel techniques that may provide a more standardized approach to quantitation of cells in tissue sections have been made possible by computerized video image analysis methods over recent years. The present study describes a novel, computer-assisted video image analysis method of quantitating immunostained cells within tissue sections, providing continuous graphical data. This technique enables the measurement of both distribution and density of cells within tissue sections. Specifically, the study considered immunoperoxidase-stained tumor infiltrating lymphocytes within breast tumor specimens, using the number of immunostained pixels within tissue sections to determine cellular density and number. Comparison was made between standard manual graded quantitation methods and video image analysis, using the same tissue sections. The study demonstrates that video image techniques and computer analysis can provide continuous data on cell density and number in immunostained tissue sections, which compares favorably with standard visual quantitation methods, and may offer an alternative.Keywords: immunostaining, video image analysis, cellular quantitation, tissue sections, breast cancer, tumor infiltrating lymphocytes

Development of a vaccine drug product requires formulation optimization to ensure that the vaccine's effectiveness is preserved upon storage throughout the shelf-life of the product. Although aluminum adjuvants have been widely used in vaccine formulations to safely and effectively potentiate an immune response, careful attention must be directed towards ensuring that the type of aluminum adjuvant does not impact the stability of the antigenic composition. PCV15 is a polysaccharide-protein conjugate vaccine comprising the pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F), each individually conjugated to the protein carrier CRM197. PCV15 was formulated with either amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHS) or aluminum phosphate adjuvant (AP) and examined for both stability and immunogenicity. Using a collection of methods to evaluate vaccine stability, it was discovered that certain PCV15 serotypes (e.g., 6A, 19A, 19F) formulated with AAHS resulted in a reduction of immunogenicity in vivo and a reduction in recoverable dose as tested by an in vitro potency assay. The same polysaccharide-protein conjugates formulated with AP were stable regarding all measures tested. Moreover, the reduction in potency of certain serotypes correlated with chemical degradation of the polysaccharide antigen caused by the aluminum adjuvant as measured by reducing polyacrylamide gel electrophoresis (SDS-PAGE), High-Pressure Size Exclusion Chromatography coupled with UV detection (HPSEC-UV) and ELISA immunoassay. This study suggests a formulation, which includes AAHS, may negatively impact the stability of a pneumococcal polysaccharide-protein conjugate vaccine that contains phosphodiester groups. This decrease in stability would likely result in a decrease in the "active" concentration of antigen dose, and herein, it is shown that such instability directly compromised vaccine immunogenicity in an animal model. The results presented in this study help to explain critical degradation mechanisms of pneumococcal polysaccharide-protein conjugate vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors report financial support was provided by Merck Sharp and Dohme LLC, a subsidiary of Merck & Co Inc. Rahway N.J. The authors report a relationship with Merck Sharp and Dohme LLC, a subsidiary of Merck & Co Inc. Rahway N.J. that includes: employment, equity or stocks, and travel reimbursement., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease but in turn have also resulted in replacement with non-vaccine serotypes. One such serotype, 35B, a multidrug resistant type, has been associated with an increase in disease. Mice were immunized intramuscularly with monovalent pneumococcal polysaccharide 35B conjugated to CRM197 containing aluminum phosphate adjuvant on days 0, 14, and 28. Pneumococcal enzyme-linked immunosorbent assay, opsonophagocytic killing assays, and competition OPA were performed for STs 35B and 29 to measure serotype-specific binding and functional antibodies. On day 52, mice were intratracheally challenged with S. pneumoniae ST29 to evaluate cross-protection. 35B-CRM197 immunized mice had binding and functional antibodies to both PnPs 35B and 29. 35B-CRM197 immunized mice were 100% protected from IT challenge with S. pneumoniae ST29 as compared to 30% survival in the naïve group. Future vaccines containing polysaccharide 35B, such as the investigational 21-valent PCV, V116, may provide cross protection against the non-vaccine serotype 29 due to structural similarity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are/were employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA and may potentially own stock and/or hold stock options in Merck & Co., Inc., Rahway, NJ, USA. PMM, WJS, JH, and JMS are named on a patent related to the V116 vaccine., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 10 12  photons s -1 ), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5-18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented., (open access.)

Invasive pneumococcal disease (IPD) is responsible for serious illnesses such as bacteremia, sepsis, meningitis, and pneumonia in young children, older adults, and persons with immunocompromising conditions and often leads to death. Although the most recent pneumococcal conjugate vaccines (PCVs) have been designed to target serotypes identified as the primary causative agents of IPD, the epidemiological landscape continues to change stressing the need to develop new PCVs. We have developed an investigational 24-valent PCV (PCV24) including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F all conjugated to CRM197 and evaluated this vaccine in adult monkeys. PCV24 was shown to be immunogenic and induced functional antibody for all vaccine serotypes. Of the serotypes common to PCV13 and V114 (PCV15), PCV24 had a similar immunogenic response with the exceptions of 23F which had higher IgG GMCs for PCV13 and V114, and 7F which had higher GMCs for PCV13. Functional antibody responses were similar for the serotypes in common between PCV24, PCV13 and V114 vaccines, with the exception of serotype 7F which was greater for PCV13. Overall, this study shows that PCV24 provided similar immunogenicity as the lower valent vaccines in adult monkeys with no apparent serotype interference. In addition, PCV24 also provided protection against pneumococcal infection in a mouse challenge model., Competing Interests: Declaration of Competing Interest All authors are/were employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and may potentially own stock and/or hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 10 12  photons s -1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 10 12  photons s -1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

Pneumococcal conjugate vaccines (PCVs) have been effective in reducing the disease burden caused by Streptococcus pneumoniae. The first licensed PCV (PCV7) was composed of capsular polysaccharides from seven serotypes. This was followed by PCV10, then PCV13, and currently there are a number of higher valency vaccines in development. As part of licensure, new vaccine iterations require assessment of immunogenicity. Since some antibodies can be non-functional, measuring functional antibodies is desirable. To meet this need, opsonophagocytic assays (OPAs) have been developed. Previous studies have shown there can be significant variations in OPA results from different laboratories. We have previously shown that standardizing OPA data using reference serum 007sp can decrease this variation. To extend this approach to additional serotypes, a panel of sera was tested by five laboratories using a multiplexed OPA for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F. Each sample was tested in five runs with 007sp tested three times in each run. Results were analyzed using a mixed effects ANOVA model. Standardization of the results significantly decreased the inter-laboratory variation for some serotypes. For serotypes 2, 8, and 11A, the variability was reduced by 40%, 45%, and 40%, respectively. For serotypes 12F, 17F, and 20B, the reductions were more modest (14%, 19%, and 24%, respectively). Standardization had little effect for the remaining serotypes. In many cases, the impact of normalization was blunted by the results from five sera that were collected after an extended post-vaccination interval. We have previously reported consensus values for 007sp for 13 serotypes, as well as the creation of a calibration serum panel ("Ewha Panel A"). Here, we report consensus values for 11 additional serotypes for 007sp and the creation of a second serum panel ("Ewha Panel B"). These consensus values will facilitate the development of next-generation PCVs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Evaluation of a pneumococcal conjugate vaccine (PCV) in an animal model provides an initial assessment of the performance of the vaccine prior to evaluation in humans. Cost, availability, study duration, cross-reactivity and applicability to humans are several factors which contribute to animal model selection. PCV15 is an investigational 15-valent PCV which includes capsular polysaccharides from pneumococcal serotypes (ST) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F all individually conjugated to cross-reactive material 197 (CRM197)., Methods: Immunogenicity of PCV15 was evaluated in infant rhesus macaques (IRM), adult New Zealand white rabbits (NZWR) and CD1 mice using multiplexed pneumococcal electrochemiluminescent (Pn ECL) assay to measure serotype-specific IgG antibodies, multiplexed opsonophagocytosis assay (MOPA) to measure serotype-specific functional antibody responses and bacterial challenge in mice to evaluate protection against a lethal dose of S. pneumoniae., Results: PCV15 was immunogenic and induced both IgG and functional antibodies to all 15 vaccine serotypes in all animal species evaluated. PCV15 also protected mice from S. pneumoniae serotype 14 intraperitoneal challenge. Opsonophagocytosis assay (OPA) titers measured from sera of human infants vaccinated with PCV15 in a Phase 2 clinical trial showed a good correlation with that observed in IRM (rs=0.69, P=0.006), a medium correlation with that of rabbits (rs=0.49, P=0.06), and no correlation with that of mice (rs=0.04, P=0.89). In contrast, there was no correlation in serum IgG levels between human infants and animal models., Conclusions: These results demonstrate that PCV15 is immunogenic across multiple animal species, with IRM and human infants showing the best correlation for OPA responses.

Background: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination., Methods: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity., Results: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge., Conclusions: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.

Pneumococcal disease continues to be a medical need even with very effective vaccines on the market. Globally, there are extensive research efforts to improve serotype coverage with novel vaccines; therefore, conducting preclinical studies in different animal models becomes essential. The work presented herein focuses on evaluating a 15-valent pneumococcal conjugate vaccine (PCV15) in mice. Initially we evaluated several doses of PCV15 in Balb/c mice. The optimal vaccine dose was determined to be 0.4μg per pneumococcal polysaccharide (PS) (0.8μg of 6B) for subsequent studies. This PS dose was chosen for PCV evaluation in mice based on antibody levels determined by multiplexed electrochemiluminescent (ECL) assays, T-cell responses following in vitro stimulation with CRM197 peptides and protection from pneumococcal challenge. We then selected four mouse strains for evaluation: Balb/c, C3H/HeN, CD1 and Swiss Webster (SW), immunized with PCV15 by either intraperitoneal (IP) or intramuscular (IM) routes. We assessed IgG responses by ECL assays and functional antibody activity by multiplexed opsonophagocytic assays (MOPA). Every mouse strain evaluated responded to all 15 serotypes contained in the vaccine. Mice tended to have lower responses to serotypes 6B, 23F and 33F. The IP route of immunization resulted in higher antibody titers for most serotypes in Balb/c, C3H and SW. CD1 mice tended to respond similarly for most serotypes, regardless of route of immunization. Similar trends were observed with the four mouse strains when evaluating functional antibody activity. Given the differences in antibody responses based on mouse strain and route of immunization, it is critical to evaluate pneumococcal vaccines in multiple animal models to determine the optimal formulation before moving to clinical trials., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chlamydia trachomatis is among the most prevalent of sexually transmitted diseases. While Chlamydia infection is a reportable event and screening has increased over time, enhanced surveillance has not resulted in a reduction in the rate of infections, and Chlamydia infections frequently recur. The development of a preventative vaccine for Chlamydia may be the only effective approach for reducing infection and the frequency of pathological outcomes. Current vaccine research efforts involve time consuming and/or invasive approaches for assessment of disease state, and MRI presents a clinically translatable method for assessing infection and related pathology both quickly and non-invasively. Longitudinal T2-weighted MRI was performed over 63 days on both control or Chlamydia muridarum challenged mice, either with or without elementary body (EB) immunization, and gross necropsy was performed on day 65. A scoring system was developed to assess the number of regions affected by Chlamydia pathology and was used to document pathology over time and at necropsy. The scoring system documented increasing incidence of pathology in the unimmunized and challenged mice (significantly greater compared to the control and EB immunized-challenged groups) by 21 days post-challenge. No differences between the unchallenged and EB immunized-challenged mice were observed. MRI scores at Day 63 were consistently higher than gross necropsy scores at Day 65, although two of the three groups of mice showed no significant differences between the two techniques. In this work we describe the application of MRI in mice for the potential evaluation of disease pathology and sequelae caused by C. muridarum infection and this technique's potential for evaluation of vaccines for Chlamydia.

Background: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a β-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model., Results: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB)., Conclusions: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.

Objectives: Evaluation of immune profiles in human breast cancer using high-sensitivity detection and analysis methods., Design: Cohort comparative analysis studies of breast tissue., Setting: Human hospital and laboratory healthcare facilities., Participants: Women over 18 years., Main Outcome Measures: Evaluation of the comparative immunophenotype of human breast carcinoma and normal breast tissues., Results: Leukocyte density and specific subgroups of lymphocytes and macrophages were generally higher in breast cancers compared to normal breast tissues. CD3, CD4, CD45RO, CD45RA(2H4), CD45 and HLA Class II (on TIL) were significantly expressed on breast tumour tissues compared with normal tissues (p < .01). Some 30% of T-cells were γδ-TCR positive, but the majority were αβ-TCR in type. CD19 (B-cell), CD14 (FMC32 and 33) and HLA Class I levels (epithelial and TIL) showed no significant differences. IL-2α receptor expression was low or absent on most TIL., Conclusions: High-sensitivity and image analysis techniques permitted accurate characterisation of the TIL infiltrate for immune profiling. Breast carcinoma showed predominance of CD4 T-cells of mainly memory phenotype. Normal breast tissues showed low leukocyte infiltration. Further correlation of these findings with clinical outcome, including survival, is proceeding with encouraging results.

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.

Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.

Objective: HIV and sexually transmitted disease (STD) surveillance patterns in Arizona suggested the need for integrated data analyses to identify trends., Methods: We compiled all HIV/AIDS cases diagnosed from 1998 to 2008 that were reported in Arizona and syphilis or gonorrhea cases diagnosed from 1998 to 2008 in Arizona. We used deterministic matching to identify individuals who were diagnosed with HIV and one or more STDs, and calculated time intervals between diagnoses., Results: Of 23,940 people with HIV/AIDS reported from 1998 to 2008, 1,899 (2.6%) had at least one syphilis or gonorrhea diagnosis from 1998 to 2008. Approximately 85% of these cases reported male-to-male sexual contact. Among males with syphilis, HIV coinfection increased from 0.5% in 1998 to 29.1% in 2008. Among males with gonorrhea, HIV coinfection increased from 2.0% in 1998 to 3.1% in 2008. Among HIV cases diagnosed from 2004 to 2008 and reported with at least one syphilis or gonorrhea diagnosis, the majority of syphilis cases (76.1%) were diagnosed at or after HIV diagnosis, whereas a majority of gonorrhea cases (54.9%) were diagnosed prior to HIV diagnosis., Conclusion: Use of the deterministic matching method identified increases in STD infections among HIV-infected people. The routine performance of this cross-matching method may be a useful tool in identifying these high-risk individuals so that targeted partner services and appropriate care referrals may be used in a timely fashion.

Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.

The incidence of invasive pneumococcal disease (IPD), caused by the approximately 91 serotypes of Streptococcus pneumoniae (PN), varies geographically and temporally as a result of changing epidemiology and vaccination patterns as well as due to regional measurement differences. Prevnar(®) (Pfizer), the first licensed pneumococcal conjugate vaccine (PCV), comprises polysaccharides (PS) from 7 serotypes conjugated to the mutant diphtheria toxin carrier protein, CRM197. In the United States and elsewhere, this vaccine has been highly efficacious in reducing the incidence of IPD caused by vaccine serotypes, however, the incidence of non-vaccine serotypes (e.g., 19A, 22F, and 33F) has increased, resulting in the need for vaccines with higher valencies. In response, 10- and 13-valent PCVs have recently been licensed. To further increase serotype coverage, we have developed a 15-valent PCV containing PS from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 and formulated on aluminum phosphate adjuvant. Vaccine immunogenicity was evaluated in infant rhesus monkeys since they, like human infants, respond poorly to unconjugated PN PS. Infant (2-3 month old) rhesus monkeys were vaccinated three times with PCV-15 or Prevnar(®) at 2 month intervals, and serotype-specific IgG antibodies were measured using a multiarray electrochemiluminescence (ECL) assay. The results indicate that antibody responses to PCV-15 and Prevnar(®) were comparable for the 7 common serotypes and that post-vaccination responses to PCV-15 were >10-fold higher than baseline for the 8 additional serotypes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 Ǻ resolution crystal structures.

The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced. Therefore, optical changes induced by X-ray exposure can be correlated with X-ray diffraction data collection. The installation of Raman spectroscopy into the beamline is also briefly reviewed. Data are now routinely generated almost simultaneously from three complementary types of experiments from the same sample. The beamline is available now to the NSLS general user population.

Animal models predictive of human disease are generally difficult to establish and reproduce. In the case of the Group A Streptococcus (GAS) bacterium, which is predominantly a human pathogen, virulence assessment in animal models is problematic. We compared a monkey colonization and pharyngitis model of infection in two macaque species to determine the optimal model for vaccine candidate evaluation. Rhesus and cynomolgus macaques were intranasally infected with a streptomycin resistant (Str(r)) GAS strain. Monkeys were monitored for body weight and temperature changes, throat swabs and sera were collected, and clinical observations were noted throughout the study. Both species exhibited oropharyngeal colonization by GAS, with rhesus macaques demonstrating a more sustained colonization through day 28 post-challenge. Veterinary observations revealed no significant differences between GAS-infected rhesus and cynomolgus macaques. Mock-infected monkeys did not exhibit clinical symptoms or GAS colonization throughout the study. ELISA results demonstrated that both rhesus and cynomolgus macaques developed anti-streptolysin-O antibody titers, with cynomolgus generating higher titers. Sera from infected monkeys produced opsonophagocytic killing and bound to the bacterium in an immunofluorescence assay. Both rhesus and cynomolgus macaques can be used for colonization studies with this GAS M3 strain, yet only mild clinical signs of pharyngitis and tonsillitis were observed., (Copyright © 2010. Published by Elsevier India Pvt Ltd.)

Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of `mystery density', i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data.

Exposure of a single crystal of the nitrite adduct of ferric myoglobin (Mb) at 100 K to high-intensity synchrotron X-ray radiation resulted in changes in the UV-vis spectrum that can be attributed to reduction of the ferric compound to the ferrous derivative. We employed correlated single-crystal spectroscopy with crystallography to further characterize this photoproduct. The 1.55 A resolution crystal structure of the photoproduct reveals retention of the O-binding mode for binding of nitrite to the iron center. The data are consistent with cryogenic generation and trapping, at 100 K, of a ferrous d(6) Mb(II)(ONO)* complex by photoreduction of the ferric precursor crystals using high-intensity X-ray radiation.

Objectives: To describe the taxonomy of methods used by pharmaceutical companies to influence physicians' nonsteroidal anti-inflammatory drug (NSAID) prescribing behaviors and to elicit physicians' perceptions of and counterbalances to these influences., Study Design: In-depth interviews analyzed using the constant comparative method of qualitative data analysis., Methods: Qualitative interviews were conducted with physicians representing various clinical specialties. Interviews were transcribed and coded inductively using grounded theory. Recruitment was stopped at 25 participants after the attainment of thematic saturation, when no new concepts emerged from ongoing analysis of consecutive interviews., Results: Physicians described a variety of influences that shaped their NSAID prescribing behaviors, including detailing and direct contact with pharmaceutical representatives, requests from patients inspired by direct-to-consumer advertisements, and marketing during medical school and residency training. Physicians described practice guidelines, peer-reviewed evidence, and opinions of local physician experts as important counterweights to pharmaceutical company influence. Local physician experts interpreted and provided context for new clinical evidence, practice guidelines, and NSAID-related marketing., Conclusions: The social and communicative strategies used by pharmaceutical companies can be adapted to improve physicians' adoption of guidelines for safer NSAID prescribing. Communicative interactions between local experts and other physicians who prescribe NSAIDs may be the critical target for future interventions to promote safer NSAID prescribing.

This manuscript chronicles the evolution of software used originally to control a diffractometer at a macromolecular crystallography beamline. The system has been augmented and rewritten. A modular and carefully organized suite of programs now handles the whole experimental environment from a single vantage point. It provides automatic logging of the experiment and communication with the user, all the way from an initial proposal to perform the work to the end of data collection. This has included construction of a relational database to organize all details of the experiment and incorporation of a robotic specimen changer to provide automation for high-throughput applications.

A package of software has been produced for the operation of the synchrotron beamline X12-C at the National Synchrotron Light Source at Brookhaven National Laboratory. Years of observing common user mistakes has enabled the production of software that reduces user errors significantly. Users of the beamline communicate with all the experimental apparatus, including both the data-collection equipment and the beamline components, including the monochromator, through an easy-to-use graphical user interface (GUI). Important features of the system are (1) its modularity, so that different underlying programs or different apparatus can be incorporated easily; (2) its ease of use, minimizing both user errors and training effort; and (3) that most of the experimental operations and parameters are logged automatically, again minimizing errors and facilitating more-or-less automatic reduction of the data. Features of the software are useful enough to have been incorporated into operations at other synchrotron beamlines.

The fine structure and immunoprotein content of the crystalloids are described in two cases of paraproteinemic crystalloidal keretopathy, both of which had clinical features thought by the referring ophthalmologists to be those of atypical lattice-type corneal dystrophy (presumably because of lattice-like lines). Most keratocytes in one case were surrounded by a mantle of densely packed tubular crystalloids. Individual tubules were annular in cross section with mean dimensions as follows: overall diameter, 29.32 nm (SD 1.26); internal diameter (core), 8.53 nm (SD 1.12); wall thickness, 10.39 nm (SD 0.85) (n = 10). Crystalloids were extracellular and found only in the corneal stroma, with none in Bowman's layer or Descemet's membrane. In the second case, the tubules had a similar distribution but formed geometric arrays with no clear relationship to, or envelopment of the keratocytes. The tubules were thin-walled, with mean dimensions as follows: overall diameter, 26.12 nm (SD 1.12); internal diameter (core), 15.46 nm (SD 1.12); wall thickness, 5.33 nm (SD 0) (n = 10). In both cases the tubules were kappa-light chain- and gamma-chain-positive. Laboratory investigations revealed the presence of two IgM-kappa paraproteins and an IgG-kappa paraprotein in the serum of the first patient. The second patient had an IgG-kappa paraproteinemia and bone marrow changes consistent with low-grade non-Hodgkin's lymphoma. These cases emphasize and extend the morphological range of corneal IgG crystalloids; the second case also demonstrates that corneal IgG crystalloids may be an early indicator of un underlying immunoproliferative disease.

Curative therapies for clinically localized prostate cancer have significant morbidity, and those patients who might be cured by aggressive management are not easily identified using current clinical information. Better biomarkers of tumor behavior need to be identified to improve clinical outcome. Chondroitin sulfate (CS), a glycosaminoglycan, may be a potentially useful biomarker as it is known to influence cell growth and differentiation and might influence malignant progression. In this study, CS was immuno-localized to the periacinar and peritumoral fibromuscular stromal tissue of nonmalignant and malignant prostates. The CS concentration was increased in the prostate tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P < 0.0001). Using Cox's univariate analysis, CS concentration, tumor grade, preoperative serum prostate-specific antigen (PSA), extracapsular extension of disease, positive surgical margins, and patient age were associated with an increased risk of PSA failure. The CS concentration was compared with the other features in two-variable regression analyses. CS and preoperative serum PSA concentrations were independent predictors of PSA failure. CS was a stronger prognostic feature than tumor grade and could predict outcome for patients with moderately differentiated tumors. Patients with a low CS concentration had significantly better progression-free survival following radical prostatectomy than patients with high levels of CS (Kaplan-Meier plot, 91% versus 49% PSA progression free at 5 years, respectively, P = 0.0038). Only postoperative pathological indices (extracapsular extension, surgical margins) were stronger predictors than CS. We conclude that measurement of prostatic CS concentrations at diagnosis may allow stratification of patients with early-stage prostate cancer for adjunctive or alternate therapies.

Tumor-infiltrating lymphocytes (TIL) are found in most human infiltrating ductal breast carcinomas. In studies of other tumors, TIL were capable of activation by IL-2, both in vitro and in vivo, to produce selective tumor cytolysis. Specific TIL-mediated tumor cytolysis in human breast tumors has recently been reported. The large numbers of TIL within human breast cancers imply that an immune response is occurring, since many of these cells express HLA class II as a late activation marker. However, the degree of early activation of the native TIL in breast tumors has not been fully investigated. Early activation markers CD69, CD43, and CD38 together with the IL-2R (CD25) and IL-2 cytokine were examined using mAbs and tissue section immunohistology. In situ hybridization was used to detect IL-2 mRNA (IL-2 mRNA) in parallel with immunohistochemical localization of IL-2. The results revealed the expression of CD69, CD43, and CD38, but markedly low CD25 (IL-2R) and IL-2 protein expression by the TIL. This strongly indicates that the TIL are an activated population of T cells that shows a deficiency in IL-2 protein and IL-2R expression despite adequate levels of IL-2 mRNA. The mechanism for apparent inhibition of IL-2 production and IL-2R expression in the presence of IL-2 mRNA is currently unclear; however, this may explain the relative anergic state of native TIL.

High-sensitivity immunoperoxidase labelling can be achieved using heavy metal enhancement of the di-amino (DAB) reaction product. For example nickel chloride combined with DAB improves the sensitivity of the method approximately 7-10 fold. This allows detection of approximately 100-200 molecules on cell surfaces. This has an obvious advantage over standard non-enhanced DAB methods which detect 1000-2000 molecules under similar conditions. This study compares standard and nickel enhanced DAB immunoperoxidase staining of cells in tissue sections using video-image analysis (VIA) measurement techniques. VIA is an objective method of evaluation of immunoperoxidase staining of separated cells and for immunostained cells in tissue sections. Nickel enhancement of the DAB reaction product reveals more positively stained cells, with higher contrast over background, giving superior qualities for VIA analysis providing continuous, reproducible, and objective data for statistical analysis.

The effects of aging and hormone manipulation on the glycosaminoglycan (GAG) content of prostatic stroma in guinea pigs were investigated. Total GAG and individual GAG classes (chondroitin, dermatan, and heparan sulfates, and hyaluronic acid) were measured biochemically in stromal extracts. Chondroitin sulfate was also measured and localized by video image analysis of immunocytochemically-stained tissue sections. The weight and total GAG (uronic acid) content of prostatic stroma increased between the ages of 2 weeks and 2 years by 7-8-fold and 4-5-fold respectively. GAG concentration per unit weight of stroma declined 4-fold during puberty and remained essentially unchanged thereafter. Similar results were obtained for each of the GAG classes. The decreases in GAG concentration were associated with a 3-fold increase in the size of the smooth muscle cells of the prostatic stroma during puberty. Hormonal control of GAG deposition in the prostatic stroma was investigated by steroid replacement in prepubertally-castrated animals. Administration of dihydrotestosterone (DHT) to castrate animals for 6 weeks resulted in significantly reduced concentrations of stromal uronic acid, compared with untreated castrate animals (P < 0.05). The GAG levels post-DHT treatment were similar to those observed after pubertal development in sham-operated control animals. Estradiol treatment had the opposite effect to that of DHT, resulting in a significantly increased concentration of uronic acid compared with castrate animals (P < 0.05). These steroid-induced changes in stromal GAG deposition were mostly contributed to by chondroitin and dermatan sulfates. Combined treatment with DHT and estradiol resulted in stromal uronic acid concentrations similar to those of animals receiving DHT alone, indicating that the effect of DHT on stromal GAG deposition is dominant over the effects of estradiol. Morphometric measurement, using computer-assisted video image analysis of a chondroitin sulfate epitope in prostatic sections stained with a monoclonal antibody (6C3), supported the biochemical data. Stereometric profiles across several sectioned glands demonstrated that chondroitin sulfate was confined to the periacinar basement membranes of the prostatic stroma in all groups except the estradiol-treated castrate animals, where the immunostaining extended from the periacinar basement membrane throughout the fibromuscular stroma. Treatment of castrate animals with estradiol alone also induced a physicochemical change in the chondroitin sulfate molecule, resulting in reduced electrophoretic mobility. In summary, this study identifies changes in the quantity, structure, and localization of chondroitin sulfate in the prostatic stroma of estradiol-treated guinea pigs. Furthermore, estradiol and DHT have opposing effects on the level of chondroitin and dermatan sulfate expression in the prostatic stroma.

To determine whether multiple features of immunohistochemical staining of the androgen receptor (AR) in prostate cancer could reliably predict androgen dependence, tumor biopsy specimens from 30 patients (stages A-D2) were stained using anti-peptide antibodies to the amino- and carboxyl-terminal of the AR. Measurements were made of the mean area and total amount (i.e., integrated optical density) of AR staining in at least 20 fields per section using a color video image analysis system, and the mean intensity of AR staining per cell and the percentage of AR positive tumor cells were derived. Video image analysis measurement identified quantitative differences in AR staining between the two antibodies, suggesting that this approach may provide a means of identifying receptor variants in prostate tumors. The AR staining measurements were analyzed by discriminant function analysis to assign individual cases to good and poor clinical outcome groups. AR staining features measured with a single antibody (e.g., amino-terminal) were sufficient to predict outcome following hormonal therapy in stage D2 patients (predictive value, 1.0), whereas all features of AR staining measured with both antibodies were required for the entire patient group (predictive value, 0.97). The principal discriminant in both patient groups contributing to the correct assignment of outcome was the mean intensity of AR staining per cell. These findings suggest that AR staining features measured by video image analysis have the potential to predict outcome in prostate cancer.

Surface molecules present in low copy numbers can be detected with high-sensitivity fluorescence flow cytometry. Many cells previously thought not to express certain molecules on their surface can now be shown to have these molecules in very low copy numbers by high-sensitivity fluorescent cytometric methods. Detection of molecules by immunoperoxidase staining methods has not previously been compared with high-sensitivity flow cytometry techniques. Computerized video image analysis (VIA) is a method that allows measurement of area and density of the immunostain chromogen reaction product in a standardized fashion analogous to flow cytometry. In this study, we compared immunoperoxidase reaction products measured by VIA methods with high-sensitivity flow cytometric measurements for cells with 10,000 down to 50 antibody molecules bound to their surfaces. Detection of 100-200 surface molecules was possible with heavy metal-enhanced immunoperoxidase methods, whereas standard immunoperoxidase methods were not as sensitive. The sensitivity of the nickel-enhanced immunoperoxidase staining method was confirmed for detection of an epitope (Tac-IL2 receptor alpha-chain) present in low numbers on the surface of peripheral blood lymphocytes.

In this study, we have examined whether tumor grade and morphometric nuclear features can predict the outcome of treatment by orchiectomy in patients with stage D2 prostate cancer. Two outcome groups based on duration of survival postorchiectomy were examined, a bad outcome group of 63 patients who died from prostate cancer within 12 months and a good outcome group of 34 patients who survived beyond 5 years. Tumors were histologically classified as well (17%), moderate (17%), or poorly differentiated (66%). Tumor grade and patient outcome were significantly associated (Mann-Whitney test; P < 0.005), with 76% of poorly differentiated tumors in the bad outcome group, and 65% of well-differentiated tumors in the good outcome group. Using discriminant function analysis, tumor grade correctly predicted outcome in 70% of cases. A statistically significant difference was also detected in nuclear shape values between the two outcome groups (P < 0.05) and histological grades (P < 0.05). Using discriminant function analysis, 51% of cases were correctly classified into outcome groups using nuclear shape factors, a figure which rose to 65% when all nuclear morphometric features were used. This demonstrates that nuclear morphometric features are of no clinical value in predicting the outcome of treatment in stage D2 disease. Furthermore, these evaluations cannot select patients who might be spared orchiectomy on the basis of a predicted poor response. However, nuclear shape and variance measurements of benign glandular epithelial cells within cancerous prostates were significantly different from those of malignant cells (P < 0.005). We conclude that, while video image analysis of prostatic nuclear shape can reliably discriminate between benign and malignant cells, nuclear morphometric features are of minimal prognostic value in men with stage D2 prostate cancer treated by androgen ablation.

Background: The histology of benign disease of the human prostate (benign prostatic hyperplasia) is heterogeneous. No other species demonstrates the same complexity, and current animal models do not appear to fully encompass the stromal and epithelial developmental changes involved in the human disease. This study describes age-related changes in the prostatic smooth muscle stroma of guinea pigs and humans, which may be pertinent to some aspects of the disease process in humans., Experimental Design: Histologic and ultrastructural changes were examined and measured in the prostates of guinea pigs during aging (2 weeks to 31 months). Similar measurements were also made in human prostatic tissues during aging and the development of benign prostatic pathology., Results: Morphometric analyses of prostates in guinea pigs and men demonstrated similar changes in stromal volume with age. The stromal volume proportion of the prostate in both species decreases at puberty due to the expansion of the epithelial cell compartment, and is followed by a progressive increase during adulthood until a maximum stromal content of approximately 75% of total tissue volume is reached at age 2 years in guinea pigs, and at age 70 years in men. The pathognomic feature of nodularity and the dramatic increase in gland size observed during the late stages of human benign prostatic disease did not occur in the guinea pig prostate. Ultrastructural analysis of guinea pig prostatic smooth muscle cells identified a progressive hypertrophy (approximately 10-fold) from prepuberty through to old age. Two-thirds of smooth muscle cells in the prostatic stroma of aging individuals of both species demonstrated perinuclear organelles (rough endoplasmic reticulum and free ribosomes) that were not present in younger individuals., Conclusions: The prominent histologic features of the guinea pig prostate during aging are increased stromal mass, significant stromal fibrosis, and occasional prostatitis. These features are frequently observed in men with clinical benign prostatic hyperplasia. The age-related increases in prostatic smooth muscle cell size and content of perinuclear organelles in the guinea pig suggest a re-activation of cellular synthetic activity. The similarity in some features of the prostatic smooth muscle stroma between aging men and guinea pigs suggests that there may be common pathophysiologic processes. We conclude that the guinea pig could be a useful model for examination of the age-related hypertrophy of the smooth muscle cell and the processes inducing reversion to a more synthetic smooth muscle cell phenotype.

We have applied a technique which was originally used for cardiac transplantation in mice, to the transplantation of human breast cancers. To our knowledge previous reports of this method for tumour xenografting have not been made. This technique has wider application for many non-vascularised tissue allografts, including for example synovial grafts for arthritis research or other tumour types in oncology research. The method is simple and reproducible within the limits of the experiments reported.

The proportion of mononuclear cells (MC) expressing some of the T cell activation markers in normal colon and colonic carcinomas was determined by immunoperoxidase staining and computer-assisted video image analysis (VIA). Tissue sections were stained with monoclonal antibodies against the T cell activation markers which included the MHC class II antigens DR, DP and DQ, interleukin 2 receptor (CD25) and the p180 (CD45RO) form of the leucocyte common antigen. The mean values for the proportion of leucocytes expressing these activation markers were 92% for DR, 61% for DP, 68% for DQ and 60% for CD45RO in tumours and 90, 62, 70 and 55% in normal tissue respectively. Few cells were stained for the IL2 receptor (mean values 7% for tumours and 5% for normal tissue) or the p220 form (CD45RA) of the leucocyte common antigen (mean values 6% for tumours and 4% for normal tissue). The proportion of MC bearing any of these markers in the normal colon was not significantly different from the matched colonic carcinomas.

A novel method of computer-assisted video image analysis (VIA) was used to determine the number of immunostained cells in tissue sections. This method permitted an accurate and objective quantification of cells of a particular phenotype. This enumeration was achieved by measuring the area stained by a test monoclonal antibody (such as the T cell marker, CD3) and comparing it with the area stained by a leukocyte common (LCA) monoclonal antibody (CD45). The proportion of T cells within the total leukocyte population in a particular tissue was then calculated. The differentiation of positive (stained) and negative (unstained) cells was uniformly maintained by setting the computer to detect a threshold for staining intensity. This enabled consistency to be maintained within a tissue section as well as between sections stained with the same antibody. In the present study, we determined the phenotype of leukocytes in colonic carcinomas by VIA and compared this with results obtained by normal visual analysis. The VIA method showed distinct advantages over normal visual analysis especially in sections which contained moderate numbers of stained cells.

The density and phenotypes of tumour-associated mononuclear cells (TAMC) in tissue sections of colonic carcinomas was determined by the technique of video image analysis (VIA). This technique allowed an accurate and objective enumeration of both total mononuclear cells (MC) in H&E stained sections and individual types of cells as revealed by immunoperoxidase staining with monoclonal antibodies in frozen sections. This enumeration allowed reliable statistical analysis of the differences between sample groups. Using this technique it was found that the density of MC in histiologically normal tissue was significantly higher than in tumour tissue. Tumours from patients with the best prognosis (stage A) had significantly higher numbers of TAMC than stage B (P less than 0.02), C (P less than 0.002) and D (P less than 0.002) tumours. The differences in the density of TAMC between tumours obtained from stage B and C and that between C and D were not significant, whereas stage B had a significantly higher TAMC density than stage D tumours (P less than 0.05). Comparing tumour differentiation, well differentiated adenocarcinomas had a significantly higher (P less than 0.05) TAMC density than poorly differentiated tumours but not moderately differentiated tumours. Moderately and poorly differentiated adenocarcinomas did not differ significantly in the density of TAMC. In examining the phenotype of these cells, it was found that T lymphocytes formed the majority of the TAMC with the CD4+ subset predominating in 28 of 29 cases. Similarly, all sections of normal colon (taken at least 4 cm away from the tumour) had more CD4+ than CD8+ cells. The proportion of the total leucocyte population that was CD3+ was comparable in normal and tumour tissue. Generally, few macrophages were present in either tumour or normal tissues. B cells (CD21%) and subset of NK cells (CD57+) were not detected in the tumours. There were no significant differences in the proportion of leucocytes which were CD4+, CD8+ and CD14+ (macrophages) between the normal colon and the tumour tissues. The types of cells in the TAMC population did not differ with tumour stage or differentiation or with the density of the TAMC itself.