Acellular Debris Research Articles

A giant gastric bezoar in Billroth II stomach: a case report on successful endoscopic removal via repeated fragmentation and dissolution technique negating the need for surgical intervention

A 76-year-old gentleman presented with anemia. He had a history of perforated duodenal ulcer six years ago, with Billroth II repair performed. A large gastric bezoar (8×6 cm2) with a clean base ulcer at the anastomotic junction was found during the initial oesophago- gastro-duodenoscopy (OGDS). Rapid urease test was negative. He presented with melena during the subsequent follow up (OGDS showed a Forrest Ib prepylori ulcer). We have successfully removed the gastric bezoar with dissolution therapy initially (injection of cokecola into the bezoar, followed by drinking 325 mL Coca-ColaTM twice daily), followed by four attempts of OGDS with endoscopic fragmentation. Histopathology reported as degenerated vegetable matter, acellular debris mixed with scattered fungal and bacterial colonies, which was compatible with bezoar. Follow-up OGDS showed complete clearance of the bezoar. Coca-ColaTM ingestion should be considered as initial treatment as it is non-invasive, and it enables further successful endoscopic fragmentation.

Visualizing the Histotripsy Process: Bubble Cloud–Cancer Cell Interactions in a Tissue-Mimicking Environment

Histotripsy is a non-invasive ultrasonic ablation method that uses cavitation to mechanically fractionate tissue into acellular debris. With a sufficient number of pulses, histotripsy can completely fractionate tissue into a liquid-appearing homogenate with no cellular structures. The location, shape and size of lesion formation closely match those of the cavitation cloud. Previous work has led to the hypothesis that the rapid expansion and collapse of histotripsy bubbles fractionate tissue by inducing large stress and strain on the tissue structures immediately adjacent to the bubbles. In the work described here, the histotripsy bulk tissue fractionation process is visualized at the cellular level for the first time using a custom-built 2-MHz transducer incorporated into a microscope stage. A layer of breast cancer cells were cultured within an optically transparent fibrin-based gel phantom to mimic cells inside a 3-D extracellular matrix. To test the hypothesis, the cellular response to single and multiple histotripsy pulses was investigated using high-speed optical imaging. Bubbles were always generated in the extracellular space, and significant cell displacement/deformation was observed for cells directly adjacent to the bubble during both bubble expansion and collapse. The largest displacements were observed during collapse for cells immediately adjacent to the bubble, with cells moving more than 150–300 μm in less than 100 μs. Cells often underwent multiple large deformations (>150% strain) over multiple pulses, resulting in the bisection of cells multiple times before complete removal. To provide theoretical support to the experimental observations, a numerical simulation was conducted using a single-bubble model, which indicated that histotripsy exerts the largest strains and cell displacements in the regions immediately adjacent to the bubble. The experimental and simulation results support our hypothesis, which helps to explain the formation of the sharp lesions formed in histotripsy therapy localized to the regions directly exposed to the bubbles.

Squamoid Cyst of Pancreatic Ducts: A Case Series Describing Novel Immunohistochemistry, Cytology, and Quantitative Cyst Fluid Chemistry

Squamoid cyst of pancreatic ducts (SCPD) is a benign pancreatic cyst often misdiagnosed preoperatively as a mucinous cyst. The histopathologic features are well described but the cytology and quantitative fluid chemistry profiles from fine-needle aspiration have not been reported. This case series discusses the cytology and cyst fluid chemistry profiles in 2 SCPDs and describes morphologic and immunohistochemical features that have not been previously reported. Fine-needle aspiration of 2 SCPDs yielded acellular debris lacking mucin or exfoliated squamous cells. Two cysts had elevated fluid carcinoembryonic antigen (CEA) and amylase levels. Positive immunohistochemical staining included cytokeratin 5/6, pCEA, synaptophysin, and chromogranin (both focal). MUC2 and MUC5AC showed negativity in all cases, while PAX8 showed negative nuclear staining. An accurate preoperative diagnosis of SCPD is potentially difficult in the setting of elevated fluid CEA levels, and acellular cytology as a mucinous cyst cannot be confidently excluded.

Case seminar: a young female with acute hyponatremia and a sellar mass

In familial cases of combined pituitary hormone deficiency the most common mutations are that of Prophet of Pit 1 (PROP1) gene. PROP1 mutations are associated with deficiencies of growth hormone, thyrotropin, prolactin, and gonadotropins (follicle-stimulating hormone and luteinizing hormone), with evolving adrenocorticotropin (ACTH) deficiency in some cases. On imaging in most patients the pituitary gland is hypoplastic, but occasionally transient pituitary enlargement is found. We report a 22-year-old female initially diagnosed at age 12 with familial hypopituitarism due to PROP1 mutation, who presented with coma and respiratory arrest (acute hyponatremia). She was urgently treated in Intensive Care Unit of Emergency Center with hypertonic saline and stress doses of hydrocortisone, which resulted in the fast increase of plasma osmolality resulting in the osmotic demyelination syndrome. Simultaneously and incidentally on computed tomography scan a large sellar and suprasellar mass were reported as possible Rathke's cleft cyst or craniopharyngioma. Once the patient was stable, ACTH deficiency was documented. She remained replaced with hydrocortisone and subsequently underwent transphenoidal surgery. The removed sellar content revealed no pituitary adenoma or pituitary cells, but only an eosinophilic, colloid-like mass, and necrotic acellular debris. Her sister with hypopituitarism had an empty sella. Genetic testing in both sisters revealed the same homozygous c.150delA mutation in PROP1 gene. Here we report two sisters with the same PROP1 mutation who presented in adulthood with different pituitary morphology, one of them with a large sellar and suprasellar mass, in which transphenoidal surgery provided an extremely rare opportunity for a histopathological analysis of the sellar content. Due to the lack of endocrine care during the transition period hypocortisolism which evolved, a consequence of PROP1 mutation, was not recognized. Empirical use of hydrocortisone in the Intensive Care in our patient with life-threatening acute hyponatremia was appropriate but because glucocorticoid therapy on its own corrects hyponatremia even after stopping hypertonic saline infusion, the risk for over-correction of hyponatremia in ACTH deficiency is high.

In vitro comminution of model renal calculi using histotripsy

Shock wave lithotripsy (SWL) suffers from the fact that it can produce residual stone fragments of significant size (>2 mm). Mechanistically, cavitation has been shown to play an important role in the reduction of such fragments to smaller debris. In this study, we assessed the feasibility of using cavitationally-based pulsed ultrasound therapy (histotripsy) to erode kidney stones. Previous work has shown that histotripsy is capable of mechanically fractionating soft tissue into fine, acellular debris. Here, we investigated the potential for translating this technology to renal calculi through the use of a commonly accepted stone model. Stone models were sonicated using a 1-MHz focused transducer, with 5-cycle pulses delivered at a rate of 1 kHz. Pulses having peak negative pressures ranging from 3 to 21 MPa were tested. Results indicate that histotripsy is capable of effectively eroding the stone model, achieving an average stone erosion rate of 26 mg/min at maximum treatment pressure; substantial stone erosion was only observed in the presence of a dense cavitational bubble cloud. Sequential sieving of residual stone fragments indicated that debris produced by histotripsy was smaller than 100 μm in size, and treatment monitoring showed that both the cavitational bubble cloud and model stone appear as hyperechoic regions on B-mode imaging. These preliminary results indicate that histotripsy shows promise in its use for stone comminution, and an optimized erosion process may provide a potential adjunct to conventional SWL procedures.

Epstein-Barr Virus-associated Diffuse Large B-cell Lymphoma Arising on Cardiac Prostheses

Primary cardiac lymphoma is extremely rare and lymphoma arising in association with prosthetic valves has been described in only 3 case reports. We describe 3 patients with diffuse large B-cell lymphoma (DLBCL) involving prosthetic heart valves and a synthetic tube graft. All 3 specimens showed shallow layering of acellular fibrinous debris over the prosthetic or synthetic materials, with tumor lymphocytes present at the luminal surface. There were frequent mitoses and abundant karyorrhectic debris. All demonstrated a nongerminal center B-cell phenotype. All 3 cases were positive for Epstein-Barr virus, but there was no staining for human herpes virus 8. There was no other evidence of distant disease at the time of diagnosis and no recurrence or dissemination occurred after surgical removal of the prosthesis, though follow-up was limited. On the basis of 2008 World Health Organization diagnostic criteria, we believe these cases should be classified as DLBCL associated with chronic inflammation. However, unlike the characteristically poor prognosis reported in this entity, we hypothesize that the disease resectability in these cardiac sites, in many cases, may allow for a better prognosis than DLBCL with chronic inflammation at other less resectable sites.

Macrophage pro-inflammatory cytokine secretion is enhanced following interaction with autologous platelets

BackgroundMacrophages are the dominant phagocyte at sites of wound healing and inflammation, and the cellular and acellular debris encountered by macrophages can have profound effects on their inflammatory profile. Following interaction with apoptotic cells, macrophages are known to switch to an anti-inflammatory phenotype. Activated platelets, however, are also a major component of inflammatory lesions and have been proposed to be pro-inflammatory mediators. In the present study, we tested the hypothesis that macrophage interaction with activated platelets results in an inflammatory response that differs from the response following phagocytosis of apoptotic cells.MethodsHuman monocyte-derived macrophages (hMDMs) were co-incubated with autologous activated platelets (AAPs) and the platelet-macrophage interaction was examined by electron microscopy and flow cytometry. The cytokines TNF-α, IL-6, and IL-23 were also measured during LPS-activated hMDM co-incubation with AAPs, which was compared to co-incubation with apoptotic lymphocytes. Cytokine secretion was also compared to platelets pre-treated with the gluococorticoid dexamethasone.ResultsMacrophages trapped and phagocytized AAPs utilizing a mechanism that was significantly inhibited by the scavenger receptor ligand fucoidan. LPS-induced macrophage secretion of TNF-α, IL-6, and IL-23 was inhibited by co-incubation with apoptotic cells, but enhanced by co-incubation with AAPs. The platelet-dependent enhancement of LPS-induced cytokines could be reversed by pre-loading the platelets with the glucocorticoid dexamethasone.ConclusionsThe interaction of human macrophages with autologous platelets results in scavenger-receptor-mediated platelet uptake and enhancement of LPS-induced cytokines. Therefore, the presence of activated platelets at sites of inflammation may exacerbate pro-inflammatory macrophage activation. The possibility of reversing macrophage activation with dexamethasone-loaded platelets is a promising therapeutic approach to treating unresolved inflammation.

Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: An analysis of 31 cases with confocal laser scanning microscopy

Introduction Patients undergoing bisphosphonate therapies may develop jaw lesions, mainly consisting in bone necrosis. No effective treatment has been proposed so far for such lesions, nor is there a uniform concept on the possible pathogenesis of this entity. Methods The study population included 31 patients with bisphosphonate-related osteonecrosis of the jaws and 10 healthy donors. All patients underwent to jaw bone biopsy and the tissue samples were divided into two parts, one of which was fixed, decalcified and routinely processed. The second part was fixed, embedded in methylmetacrylate, cut and stained with methylene blue and basic fuchsine. All samples were subjected to light and confocal microscopic examination and to histomorphometric analyses to evaluate differences in bone structure between the two samples groups. Results Three main histological patterns were identified in ONJ patients: 1 — Areas with active acute inflammation, characterized by predominance of soft tissues, inflammatory infiltrate, acellular necrotic debris, thin-walled and dilated blood vessel, intensely basophilic bone spiculae with scalloped borders showing prominent bone resorption. 2 — Areas characterized by predominance of bony structures showing wide acellular necrotic sequestra and large, scalloped Haversian canals containing inflammatory cells. 3 — Non-necrotic areas contained larger amounts of bone, showing increased trabecular thickness, inter-osteonic bone deposition and smaller and fewer Haversian canals. These differences were more evident after comparing the histomorphometrical data of samples from ONJ patients with controls. Also, lamellar bone from treated patients was composed of bigger osteones containing larger osteocytes. Two different types of newly-formed woven bone, mainly showing centrifugal spatial orientation, were easily detectable in these areas. Osteoclast-like cells detected in inflammatory areas from treated patients were small and contained few nuclei, but they were rare to absent in non-necrotic bone from the same patients. Conclusions These features point at a peculiar process of bone remodeling in patients undergoing bisphosphonate therapy, which showed scarce osteoclastic activity and subsequent deposition of newly-formed bone. The latter would be made up of thicker bone structures supplied by fewer blood vessels. Consequently, in case of increased metabolic requests, this modified bone would not be supported by adequate blood supply, thus leading to necrosis and superinfection.

Les grains de milium de l’enfant

Steatocystoma multiplex is a rare condition characterized by multiple subcutaneous nodules classically located on the anterior chest. Diagnosis usually is made clinically or by biopsy. The lesions may be numerous, and surgical treatment may not be practical or the results may be unsatisfactory. Three patients with multiple subcutaneous nodules underwent fine needle aspiration, which produced smears with acellular debris. A diagnosis was possible in the first patient because of close collaboration with a dermatologist. The lesions in the other patients were recognized clinically and were confirmed cytologically by fine needle aspiration. A syringe holder allows the use of thin needles (22-gauge) to aspirate very small lesions (4 to 5 mm). Fine needle aspiration may provide a useful alternative to surgical methods in the treatment of these lesions because it is inexpensive, well-tolerated by the patients, and has no associated morbidity.

Amebiasis

Amebiasis is defined as infection with Entamoeba histolytica, regardless of associated symptomatology. In resource-rich nations, this parasitic protozoan is seen primarily in travelers to and emigrants from endemic areas. Infections range from asymptomatic colonization to amebic colitis and life-threatening abscesses. Importantly, disease may occur months to years after exposure. Although E histolytica was previously thought to infect 10% of the world's population, 2 morphologically identical but genetically distinct and apparently nonpathogenic Entamoeba species are now recognized as causing most asymptomatic cases. To avoid unnecessary and possibly harmful therapies, clinicians should follow the diagnostic and treatment guidelines of the World Health Organization.

Pulsed Cavitational Ultrasound: A Noninvasive Technology for Controlled Tissue Ablation (Histotripsy) in the Rabbit Kidney

The optimal minimally invasive treatment for small renal masses continues to evolve. Current ablative technologies rely on thermal mechanisms for tissue destruction. However, the creation of precise lesions is limited by inhomogeneous heating/cooling due to tissue variability, perfusion effects and tissue charring. We hypothesized that nonthermal mechanical effects of ultrasound (cavitation) can be used to progressively homogenize tissue in controlled fashion with predictable results. We developed a focused annular array ultrasound system capable of delivering high intensity (greater than 20 kW/cm) short pulses (20 microseconds) of energy to a target volume. This system operates at a repetition frequency of 100 Hz, resulting in a low time averaged power output (approximately 5 W total acoustic output). Following approval from the institutional animal care committee a series of transcutaneous ablations were performed in the normal kidneys of 10 rabbits. Lesions created with a small number of pulses (10 or 100) produced scattered areas of damage characterized by focal hemorrhage and small areas of cellular injury in the targeted volume. Lesions created with greater numbers of pulses (1,000 or 10,000) demonstrated complete destruction of the targeted volume. Gross examination revealed that lesions contained a liquefied core with smooth walls and sharply demarcated boundaries. Histological examination demonstrated extensive areas of acellular debris surrounded by a narrow margin of cellular injury. This pulsed cavitational ultrasound system is capable of transcutaneous nonthermal destruction of renal tissue. Refinement of this technology for noninvasive ablation of small renal masses is currently under way.

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1β. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.

Cellular Destruction Following Transmyocardial Laser Revascularization (TMR)

During transmyocardial revascularization, cellular destruction of cardiomyocytes occurs as a result of the high-energy laser. However, the features of myocardial cellular destruction are unclear. The present study was undertaken to examine the structural characteristics of cell death in the myocardium following transmyocardial revascularization. Myocardial specimens from 3 male patients who had died within 11 days following laser revascularization were collected within 1 h of death and were analyzed by immunohistochemistry and electron microscopy. For immunohistochemistry, antibodies to pro-apoptotic proteins CPP32 and BAX were used. Immunohistochemical examination demonstrated the presence of cells expressing both CPP32 and BAX along the laser channel. Electron microscopic analysis revealed that the lining surface of laser channels consisted of condensed acellular debris and dead cells. No endothelialization of channels was noted. The lumen of laser channels were surrounded by a rim of acellular debris with several outer concentric rims of cardiomyocytes showing features of cellular destruction. The present study identified features of both necrotic and apoptotic cellular death following laser revascularization.

Particle disease: Cytopathologic findings of an unusual case

Particle disease is a rare lesion that results from an inflammatory response due to wear debris-induced osteolysis following arthroplasty. Particles resulting from the wear debris cause macrophage activation and phagocytosis. Particle disease often leads to joint loosening and implant failure. Radiologically, it often results in a well-defined osteolytic lesion-mimicking tumor. A 68-year-old man who presented with chronic hip pain following total hip replacement was studied by fine-needle aspiration. An ultrasound-guided aspiration revealed hypercellular smears consisting predominantly of proliferating mesenchymal cells, foamy macrophages, inflammatory cells, and background acellular debris. Numerous multinucleated giant cells were observed as well. Differential diagnosis includes primary or metastatic clear-cell tumors.

BILATERAL KERATINIZING DESQUAMATIVE SQUAMOUS METAPLASIA OF THE UPPER URINARY TRACT

A 58-year-old man presented with leR renal colic. He had a 10-year history of recurrent bilateral pain with passage of multiple stones, the composition of which had never been analyzed. At age 8 years he was diagnosed with primary pulmonary and osteoarticular tuberculosis affecting the spine and at age 20 years he underwent lumbar arthrodesis with bone graft. Creatinine was 2.1 mg./dl. (normal 0.5 to 1.3) with moderate leukocytosie. Urinalysis showed sterile pyuria and abundant acellular debris, and radiography demonstrated diffise bilateral renal calcification. Sonography revealed a cystic mass in the right kidney with a prominent acoustic shadow and a large heterogeneous left renal mass with adjacent calcification. Excretory urography showed severely delayed excretory function with loss of left renal structure and a polycyclic circumscribed filling defect occupying the pelvis, calices and proximal ureter as well as caliceal distortion and a large cavern in the right kidney (fig. 1). Based on these findings and the history renal tuberculosis was strongly suspected. Extensive bilateral calcification, osteoarticular and pulmonary stigmata, sterile pyuria and occasional fever supported the diagnosis. A Mantow test was positive but smears and cultures for acid-fast bacilli were negative. Computerized tomography confirmed severe leR renal disease and did not rule out a possible upper tract tumor. Urine cytology revealed small groups of well differentiated transitional cells and diffuse normal keratotic scales interpreted at that time as contamination. Left nephroureterectomy was performed. Grossly a graywhite, leathery, choral-like structure occupied the pelvis, calices and ureter. Microscopy revealed renal atrophy, the absence of urothelium and complete substitution by well differentiated, mature, nondysplastic stratified squamous epithelium (fig. 2). There was no evidence of a granulomatous response, caseous necrosis or ulcerative cavernous lesion to suggest a mycobacterial infection. Postoperatively the passage of desquamated epithelium resembling SOR stones or gritty flakes confirmed bilateral disease. Ureteroscopy revealed whitish plaques and incrustations in the distal ureter and renal pelvie. A keratin plug was extracted and histological examination again revealed keratinizing desquamative squamous metaplasia. Three years later the condition remains under control. The patient

Association of human papillomavirus with malignant and premalignant lesions of the uterine endometrium

The possible association of human papillomavirus (HPV) with endometrial hyperplasia and endometrial adenocarcinoma was investigated. DNA from frozen tissues of 30 endometrioid carcinomas of Japanese patients was tested for HPV DNA by Southern blot hybridization analysis. Screening with HPV type 58 probe under low stringency conditions showed the presence of HPV DNA in two of 30 endometrioid carcinomas. High stringency hybridization identified HPV type 16 in the two positive specimens. The presence of HPV was further analyzed by polymerase chain reaction (PCR)-Southern blot analysis of DNA from archival tissue blocks of the initial 30 endometrioid carcinomas as well as an additional 17 endometrioid carcinomas and 13 atypical hyperplasias of the endometrium from Japan and 38 endometrioid carcinomas from the United States. Polymerase chain reaction amplification using type 16-specific HPV primers for a portion of the E6 open reading frame was positive in six of 47 (13%) endometrioid carcinomas from Japan, including two in which HPV 16 was not detected by Southern blot analysis and two of 38 (5%) endometrioid carcinomas from the United States. Polymerase chain reaction amplification using Ll consensus sequence primers was positive for HPV in two of 13 (15%) endometrial hyperplasias, 13 of 47 (28%) endometrioid carcinomas from Japan, and six of 38 (16%) endometrioid carcinomas from the United States. Slot blot hybridization identified HPV type 16 in seven of the L1 PCR products, including all but one specimen testing positive for HPV type, 16 using E6 type specific primers. In situ hybridization was positive for HPVs 16/18 in glandular epithelial tumor cells in six of the PCRpositive specimens. An additional specimen showed staining for HPVs 16/18 in acellular luminal debris in association with squamous metaplasia of the tumor, but staining was negative in the glandular cells of the tumor. Human papillomavirus was not detected by in situ hybridization in the remaining specimen, which was PCR positive for HPV 16. In situ hybridization was weakly positive for HPVs 31/33/ 35 in one specimen and was weakly positive for HPVs 6/11 in benign endometrial epithelial cells but not in tumor cells of another specimen that tested positive for HPV by Ll PCR. Two dimensional gel electrophoresis performed on two specimens showed that HPV DNAs were integrated into cellular DNA with no episomal coexistence. These findings suggest that HPV, especially HPV 16, may play an etiologic role in a fraction of endometrioid adenocarcinomas.

A simple electronic volume cell sorter for clonogenicity assays.

A single-parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to approximately 2,000 per second with greater than 80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including 1) precise cell plating for low-dose survival studies, 2) separation of cells into age compartments, and 3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.

Intravascular macrophages in lungs of pigs infected with Haemophilus pleuropneumoniae.

Pigs were inoculated intratracheally with a virulent or an avirulent isolate of Haemophilus pleuropneumoniae serotype 5 and sacrificed during the first 24 hours post-inoculation. Intravascular macrophages were examined by electron microscopic and morphometric techniques. Samples of lung were taken from regions with no macroscopic lesions (Zone 0), 2.5 to 3.0 cm from lesions (Zone 1), and from the immediate edge of lesions (Zone 2). Those pigs inoculated with the avirulent isolate did not develop lesions. Pigs given the virulent isolate consistently developed necrohemorrhagic lesions in the dorsolateral aspect of the caudal and middle lung lobes. Relative volumes of intravascular macrophages in Zones 1 and 2 increased with increased time post-inoculation; in pigs given the avirulent isolate, intravascular macrophage volume decreased with increased time post-inoculation. Cytoplasmic volume to nuclear volume ratios for macrophages in Zone 2 from pigs with necrohemorrhagic lesions progressively increased with increased time post-inoculation. Enlarged intravascular macrophages had large nuclei, prominent nucleoli, and abundant cytoplasm. Increased cytoplasmic volume was the result of increased numbers of lysosomes, phagosomes, rough and smooth endoplasmic reticulum, and large Golgi complexes. Pigs inoculated with the virulent bacteria had IV macrophages with large phagosomes that contained necrotic cell debris and fibrin. Macrophages with phagosomes were more frequent in the 6-, 9-, and 24-hour sample periods of pigs with lesions than in any other group. Intercellular adhesion plaques (ICAP) were present between IV macrophages and subjacent endothelial cells. ICAP's increased in length with increased time post-inoculation in Zones 1 and 2 from pigs with necrohemorrhagic lesions. In later sample periods, multiple closely associated and interlacing IV macrophages formed a discontinuous layer over endothelial cells in Zone 2 samples near necrohemorrhagic lesions. These results suggest that the intravascular macrophage population changes from immature macrophages to mature macrophages or immature epithelioid cells within 24 hours after inhalation of a virulent Haemophilus pleuropneumoniae. Furthermore, intravascular macrophages likely function to clear cellular and acellular debris from the blood in pneumonic conditions.

Retention of intact cells on bronchoscopy brushes after cleaning.

Reusable nylon bristle bronchoscopy brushes were evaluated to assess whether cytologically recognizable cells could be retained on the bristles after conventional cleaning techniques. Twenty brushes were used during bronchoscopy; 5 brushes were inserted directly into freshly resected neoplasms. The brushes were then scrubbed and rinsed in chlorhexidine gluconate, iodine, and alcohol, and air dried. Several days later fresh cytologic specimens were prepared from the apparently clean brushes. Eighty percent of the cytologic specimens exhibited strands of mucus, most containing well-preserved alveolar macrophages, polymorphonuclear leukocytes, and ciliated epithelial cells. Although no malignant cells were retained, it is clear that conventional cleaning techniques are inadequate for the removal of cells and acellular debris. Consideration should be given to immersing brushes in acetylcysteine or employing disposable brushes.

CELL LOSS FROM THREE ESTABLISHED LINES OF THE C3H MOUSE MAMMARY TUMOR: A COMPARISON OF THE 125I‐UdR AND THE 3H‐TdR‐AUTORADIOGRAPHIC METHODS

The 125I-UdR method for measuring cell loss from solid tumors has been reevaluated. The rate of tumor cell loss from three established lines (S 102F, S102S and Slow) of the C3H mouse mammary tumor was determined by the 125I-UdR method and the results were compared to the estimates for cell loss as determined by the combined approach of cellular 3H-TdR autoradiography and volumetric growth-rate determinations. This detailed comparison shows that the two methods complement each other but cannot substitute for one another because they give different quantitative information. The combined approach measures the flow of viable cells, as determined morphologically, from the proliferating compartment to the quiescent comparment, the quiescent compartment out of the tumor, etc., but does not evaluate the flow of degenerate cells or acellular (necrotic) debris. In contrast, the 125I-UdR method indicates the net flow of intact cells and/or dead cells as well as debris from the tumor as the 125I-labeled material is lost from the tumor, but gives limited internal information. thus, depending on the specific experiment, an investigator could choose one or the other of the methods to answer the question. Perhaps both would be desirable at times; however, in most cases, one could not substitute one method for the other. The data from the Slow tumors also indicate that in certain tumors, the quantitative information from the 125I-UdR method may be quite limited, i.e. the confidence limits within an experiment as well as the replication error between experiments may be high.