Comparison of bone healing, as assessed by computed tomography, following tibial tuberosity advancement in dogs with and without autogenous cancellous bone grafts.

AIMS: To objectively compare measures of bone healing, using computed tomography (CT) in dogs following bilateral tibial tuberosity advancement (TTA), between tibiae treated with and without autogenous cancellous bone grafts. METHODS: Ten dogs with bilateral cranial cruciate ligament disease requiring surgical stabilisation were prospectively recruited to undergo single-session bilateral TTA, with only one, randomly assigned, tibia receiving bone graft in the osteotomy deficit. Bone healing at the osteotomy site was assessed using CT performed 38-70 days post-operatively. CT images were evaluated using both objective measurements of osseous bridging and subjective evaluation by six radiologists. Repeated measures ANOVA was used to compare the objective outcomes between the grafted and non-grafted tibiae. RESULTS: The mean percentage of the osteotomy deficit bridged at the lateral cortex was greater in grafted (77.6, SD 35.2%) compared to non-grafted (63.0, SD 36.5%) tibiae (p=0.001), but did not differ at the medial cortex (p=0.1). The mean minimum callus width was greater in grafted (7.2, SD 3.3 mm) compared to non-grafted (3.6, SD 2.9 mm) tibiae (p<0.001). There was no difference in mean attenuation (measured in Hounsfield units) of the callus between grafted and non-grafted tibiae (p=0.5). The grafted tibia was deemed to have superior bone healing in 50/60 subjective assessments made by radiologists. CONCLUSIONS: Superior osseous bridging was detected by CT analysis following TTA using autogenous cancellous bone grafts compared with no graft. This was shown by greater bridging percentage at the lateral cortex and formation of a broader callus. Qualitative assessments made by six radiologists also supported the conclusion that bone healing was improved by use of autogenous cancellous bone graft. CT was a useful method for assessing evidence of bone healing following TTA. CLINICAL RELEVANCE: These findings justify the application of autogenous cancellous bone graft to augment healing following TTA in dogs.

Potential mechanisms for lung fibrosis associated with COVID-19 infection.

Pulmonary fibrosis is a sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection that currently lacks effective preventative or therapeutic measures. Post-viral lung fibrosis due to SARS-CoV-2 has been shown to be progressive on selected patients using imaging studies. Persistent infiltration of macrophages and monocytes, a main feature of SARS-CoV-2 pulmonary fibrosis, and long-lived circulating inflammatory monocytes might be driving factors promoting the profibrotic milieu in the lung. The upstream signal(s) that regulates the presence of these immune cells (despite complete viral clearance) remains to be explored. Current data indicate that much of the stimulating signals are localized in the lungs. However, an ongoing low-grade systemic inflammation in long Coronavirus Disease 2019 (COVID-19) symptoms suggests that certain non-pulmonary regulators such as epigenetic changes in hematopoietic stem cells might be critical to the chronic inflammatory response. Since nearly one-third of the world population have been infected, a timely understanding of the underlying pathogenesis leading to tissue remodeling is required. Herein, we review the potential pathogenic mechanisms driving lung fibrosis following SARS-CoV-2 infection based upon available studies and our preliminary findings (Graphical abstract).

New Insights on Diagnostic Reproducibility of Biphasic Mesotheliomas: A Multi-Institutional Evaluation by the International Mesothelioma Panel From the MESOPATH Reference Center.

INTRODUCTION: The 2015 WHO classification of tumors categorized malignant mesothelioma into epithelioid, biphasic (BMM), and sarcomatoid (SMM) for prognostic relevance and treatment decisions. The survival of BMM is suspected to correlate with the amount of the sarcomatoid component. The criteria for a sarcomatoid component and the interobserver variability between pathologists for identifying this component are not well described. In ambiguous cases, a "transitional" (TMM) subtype has been proposed but was not accepted as a specific subtype in the 2015 WHO classification. The aims of this study were to evaluate the interobserver agreement in the diagnosis of BMM, to determine the nature and the significance of TMM subtype, and to relate the percentage of sarcomatoid component with survival. The value of staining for BRCA-1-associated protein (BAP1) and CDKN2A(p16) fluorescence in situ hybridization (FISH) were also assessed with respect to each of the tumoral components. METHODS: The study was conducted by the International Mesothelioma Panel supported by the French National Cancer Institute, the network of rare cancer (EURACAN) and in collaboration with the International Association for the Study of Lung Cancer (IASLC). The patient cases include a random group of 42 surgical biopsy samples diagnosed as BMM with evaluation of SMM component by the French Panel of MESOPATH experts was selected from the total series of 971 BMM cases collected from 1998 to 2016. Fourteen international pathologists with expertise in mesothelioma reviewed digitally scanned slides (hematoxylin and eosin - stained and pan-cytokeratin) without knowledge of prior diagnosis or outcome. Cases with at least 7 of 14 pathologists recognizing TMM features were selected as a TMM group. Demographic, clinical, histopathologic, treatment, and follow-up data were retrieved from the MESOBANK database. BAP1 (clone C-4) loss and CDKN2A(p16) homozygous deletion (HD) were assessed by immunohistochemistry (IHC) and FISH, respectively. Kappa statistics were applied for interobserver agreement and multivariate analysis with Cox regression adjusted for age and gender was performed for survival analysis. RESULTS: The 14 panelists recorded a total of 544 diagnoses. The interobserver correlation was moderate (weighted Kappa = 0.45). Of the cases originally classified as BMM by MESOPATH, the reviewers agreed in 71% of cases (385 of 544 opinions), with cases classified as pure epithelioid in 17% (93 of 544), and pure sarcomatoid in 12% (66 of 544 opinions). Diagnosis of BMM was made on morphology or IHC alone in 23% of the cases and with additional assessment of IHC in 77% (402 of 544). The median overall survival (OS) of the 42 BMM cases was 8 months. The OS for BMM was significantly different from SMM and epithelioid malignant mesothelioma (p < 0.0001). In BMM, a sarcomatoid component of less than 80% correlated with a better survival (p = 0.02). There was a significant difference in survival between BMM with TMM showing a median survival at 6 months compared to 12 months for those without TMM (p < 0.0001). BAP1 loss was observed in 50% (21 of 42) of the total cases and in both components in 26%. We also compared the TMM group to that of more aggressive patterns of epithelioid subtypes of mesothelioma (solid and pleomorphic of our large MESOPATH cohort). The curve of transitional type was persistently close to the OS curve of the sarcomatoid component. The group of sarcomatoid, transitional, and pleomorphic mesothelioma were very close to each other. We then considered the contribution of BAP1 immunostaining and loss of CDKN2A(p16) by FISH. BAP1 loss was observed in 50% (21 of 41) of the total cases and in both component in 27% of the cases (11 of 41). There was no significant difference in BAP1 loss between the TMM and non-TMM groups. HD CDKN2A(p16) was detected in 74% of the total cases with no significant difference between the TMM and non-TMM groups. In multivariate analysis, TMM morphology was an indicator of poor prognosis with a hazard ratio = 3.2; 95% confidence interval: 1.6 - 8.0; and p = 0.003 even when compared to the presence of HD CDKN2A(p16) on sarcomatoid component (hazard ratio = 4.5; 95% confidence interval: 1.2 - 16.3, p = 0.02). CONCLUSIONS: The interobserver concordance among the international mesothelioma and French mesothelioma panel suggests clinical utility for an updated definition of biphasic mesothelioma that allows better stratification of patients into risk groups for treatment decisions, systemic anticancer therapy, or selection for surgery or palliation. We also have shown the usefulness of FISH detection of CDKN2A(p16) HD compared to BAP1 loss on the spindle cell component for the separation in ambiguous cases between benign florid stromal reaction from true sarcomatoid component of biphasic mesothelioma. Taken together our results further validate the concept of transitional pattern as a poor prognostic indicator.

Negative pressure wound therapy using a portable single-use device for free skin grafts on the distal extremity in seven dogs.

OBJECTIVE: Retrospective study to describe clinical experience with a portable single-use negative pressure wound therapy device after application of full-thickness meshed skin grafts to wounds on the distal extremities of seven dogs. METHODS: Seven dogs were treated with portable NPWT after receiving skin grafts; six as the result of tumour resection and one for traumatic injury. Medical records were reviewed and data recorded on patient signalment, cause and location of wound, surgical technique, application and maintenance of portable NPWT, graft survival and outcome, and complications encountered with the system. CLINICAL OUTCOMES: NPWT was provided for between 4 and 7 days. Five patients were discharged from hospital during the treatment period. Application and maintenance of the portable device was technically easy and no major complications were encountered. Minor complications consisted of fluid accumulation in the evacuation tubing. All dogs achieved 100% graft survival. CONCLUSIONS: Application and maintenance of the portable device was technically straightforward. All dogs receiving portable NPWT after transfer of a free skin graft to the distal extremity had a successful outcome.

A stable prostacyclin analog, beraprost sodium, attenuates platelet accumulation and preservation-reperfusion injury of isografts in a rat model of lung transplantation.

BACKGROUND: Recent studies have shown that the extent of platelet accumulation in the vasculature of transplanted organs correlates with the degree of preservation-reperfusion injury. In this study, we examined the effect of a stable prostacyclin analog, beraprost sodium, which possesses potent antiplatelet activity, on parameters of platelet accumulation and preservation-reperfusion injury of isografts in a rat model of lung transplantation. METHODS: The heart-lung blocks of donor rats were flushed with and preserved in modified Euro-Collins solution at 4 degrees C for 6 hr or 24 hr. The left lung was transplanted into recipient rats and reperfused for 1 hr. Lung injury was evaluated by the pulmonary blood flow ratios to the lung isografts, the weight gain of the isografts, and histological examination. Small portions of the lung isografts were excised and stained with an antibody specific for rat platelets. A scoring system was developed to semiquantitate the intensity of antibody staining (score 0-4). The recipient rats received oral administration of beraprost sodium (0.3 mg/kg) before lung transplantation. Control animals received no beraprost sodium. RESULTS: In the 6-hr preservation study, administration of beraprost sodium significantly reduced the score for platelet accumulation (1.8+/-0.4 vs. 3.3+/-0.5, P<0.01). This observation was accompanied by a significantly decreased degree of preservation-reperfusion injury as evidenced by an increased blood flow ratio (13.7+/-2.6% vs. 4.5+/-3.6%, P<0.01) and a reduced weight gain (0.7+/-0.2 g vs. 1.1+/-0.2 g, P<0.01). Histological examination revealed severe capillary congestion in three of six cases in the control group, while no capillary congestion was observed in the beraprost group. In the 24-hr preservation study, no differences were seen in platelet accumulation scores or parameters of lung injury. CONCLUSION: Beraprost sodium, an antiplatelet agent, reduces platelet accumulation and preservation-reperfusion injury of lung transplants at 6 hr in this rat isograft model.

Assessment of pathological changes associated with chronic allograft rejection and tolerance in two experimental models of rat lung transplantation.

Lung transplantation is now routinely performed for a wide range of end-stage cardiopulmonary disorders. Despite overcoming the problems associated with early acute rejection, chronic rejection (CR) in the form of obliterative bronchiolitis has emerged as the primary cause of late graft loss. The mechanisms involved in the development of CR of lung allografts are poorly understood, and no effective therapy is currently available. To better understand the pathological events associated with CR and tolerance, we examined two models of lung allograft rejection established in our laboratory. First, we exchanged left lung allografts between moderately histoincompatible inbred rat strains (WKY-->F344: n = 42 and F344-->WKY: n = 40). The WKY-->F344 model was previously shown to develop spontaneous tolerance, while the converse model (F344-->WKY) showed persistent acute rejection. The purpose of this investigation was to assess histopathological changes associated with long-term grafts left in place up to 140 days after transplant. To confirm that tolerance had developed, skin-grafting experiments were performed. Five skin grafts from each strain were placed on lung allograft recipients on day 35 after transplant and skin allograft survival was assessed and compared with controls. Acute rejection (AR) was graded histologically (stage O-IV) and the pathologic intensity of inflammation and CR were graded (0-4: 0 = 0%, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%) on percentage of involvement with the following categories being examined: (a) lymphocytic infiltration (perivascular, peribronchial, and peribronchiolar) and (b) vasculitis, edema, hemorrhage, and necrosis. Finally, chronic rejection was diagnosed by the presence of intimal hyperplasia, interstitial fibrosis, peribronchiolar fibrosis, bronchiolitis obliterans, and bronchiectasis. The WKY-->F344 animals showed progressive AR (stage III, day 21). Thereafter, the AR subsided spontaneously and was stage 0 on day 140. There were no signs of CR in these animals. In the F344-->WKY model, the AR progressed up to stage III-IV (day 21) and maintained for several weeks at stage III. Thereafter, pictures of the lungs showed CR on days 49, 70, and 98. There were significant differences between the two models during the chronic phase, such as interstitial fibrosis (0 +/- 0 vs. 1.8 +/- 1.3, P < 0.005), peribronchiolar fibrosis (0 +/- 0 vs. 3.6 +/- 0.55, P < 0.01), vasculitis (0.2 +/- 0.45 vs. 2.0 +/- 0, P < 0.008), and intimal hyperplasia (0.2 +/- 0.45 vs. 2.6 +/- 0.9, P < 0.008).(ABSTRACT TRUNCATED AT 250 WORDS)

Prolongation of skin allograft survival is associated with reduced Th1 cytokine responses in the WKY-->F344 rat model.

BACKGROUND: We have reported previously that F344 rats develop a spontaneous tolerance to WKY lung allografts and show long-term retention of donor-specific skin grafts placed 35 days after lung transplantation. In this study, we investigated the immunologic mechanisms that may be responsible for the prolonged skin graft survival in animals tolerized with lung allografts. METHODS: In the rejection group, WKY skin grafts were placed on normal F344 rats, whereas, in the tolerance group, the skin grafts were placed on F344 rats that had received a WKY lung transplant 35 days before skin grafting. Th1 (interleukin [IL]-2 and interferon-gamma [IFN-gamma]) and Th2 (IL-4 and IL-10) cytokine as well as transforming growth factor-beta1 mRNA expression in skin grafts and in draining lymph nodes were determined by reverse transcription-polymerase chain reaction. Macrophage and lymphocyte infiltration in skin grafts and the number of Langerhans cells in epidermal sheets of the grafts were examined by immunohistochemistry. RESULTS: IL-2 and IFN-gamma mRNA expression was significantly decreased in both the skin grafts and the draining lymph nodes of the tolerance group, compared to the rejection group, whereas IL-10 and transforming growth factor-beta1 mRNA expression was similar in both groups and IL-4 mRNA was rarely detected. Decreased and delayed CD8+, macrophage, and natural killer cell infiltration in the skin grafts from the tolerance group was also detected. Similar reduction in the number of Langerhans cells in the epidermis of the grafts from both groups was seen on day 1 after skin grafting, and thereafter the number remained stable in both groups. CONCLUSIONS: Reduced expression of Th1 cytokines and decreased infiltration of CD8+ cells, macrophages, and natural killer cells in the skin grafts may be responsible for prolongation of skin graft survival in the tolerance group.

Accumulation of platelets in rat syngeneic lung transplants: a potential factor responsible for preservation-reperfusion injury.

BACKGROUND: Platelets are known to play an important role in the pathogenesis of adult respiratory distress syndrome as well as preservation-reperfusion injury of liver allografts. However, the role of platelets in pulmonary preservation-reperfusion injury is unknown. In this study, we examined whether the extent of platelet accumulation in the preserved and subsequently reperfused lungs correlated with the degree of preservation-reperfusion injury in a rat lung isotransplant model. METHODS: Heart-lung blocks from donor rats were flushed with and preserved in modified Euro-Collins solution at 4 degrees C for 0 hr (n=5), 6 hr (n=6), and 24 hr (n=6). The left lung was divided from the heart-lung block, transplanted into the recipient rat, and reperfused for 1 hr. Lung injury was evaluated by the left-to-right pulmonary blood flow ratio, the weight gain of the isograft, and the scores for histological categories of lung injury (intra-alveolar edema, intra-alveolar hemorrhage, and capillary congestion). Small portions of the lung isograft were excised and stained with an antibody specific for rat platelets. A scoring system was developed to semiquantitate the intensity of antibody staining in isografts. RESULTS: Lung isografts were injured and platelets accumulated in the capillaries in proportion to the length of preservation endured before transplantation. The extent of platelet accumulation evaluated by our morphological scoring system correlated significantly with the degree of lung injury assessed by the blood flow ratio (P<0.001), the weight gain (P<0.001), and the histological scores for intra-alveolar hemorrhage (P<0.05) and for capillary congestion (P<0.001). CONCLUSIONS: The results of this study suggest that platelet accumulation is a potential factor responsible for preservation-reperfusion injury of lung isografts in the rat.

Soluble CTLA4Ig modifies parameters of acute inflammation in rat lung allograft rejection without altering lymphocytic infiltration or transcription of key cytokines.

The efficacy of CTLA4Ig in blocking immune activation and allograft rejection (AR) was tested in an aggressive and rapid model of rat lung AR (Brown Norway [BN]-->Lewis [LEW]). CTLA4Ig is a recombinant soluble protein that binds with high affinity to rat B7/BB1 and other surface molecules on APCs, subsequently blocking the binding of B7/BB1 to CD28/CTLA4 on T cells. This interrupts the costimulatory pathway critical for complete T cell activation and completion of the AR process. Left single-lung transplants were performed between BN-->Lew. Five allograft recipients were examined in each group. At transplantation, animals received 250 micrograms of CTLA4Ig or 250 micrograms of control Ig intraperitoneally daily until sacrifice. Animals were sacrificed on days 2, 4, and 7 after transplant. Control (BN-->Lew) grafts show irreversible rejection by day 7. Syngeneic (Lew-->Lew) grafts show no AR on day 7. AR episodes were graded histologically (stages 0-IV) and pathologic intensity of inflammation was graded on percentage of involvement. Cytokine transcript levels were measured in control and CTLA4Ig-treated animals (n = 5 in each group) on day 7 using reverse transcriptase polymerase chain reaction techniques. The most profound differences were found on day 7 after transplant. The degree of lymphocytic infiltration was greater in the CTLA4Ig group (perivascular: 4 +/- 0 vs. 2.6 +/- 0.6, peribronchial: 4 +/- 0 vs. 2.2 +/- 0.4, and peribronchiolar: 3.6 +/- 0.5 vs. 2 +/- 0.3, P < 0.01). However, in striking contrast, the stage of AR (3 +/- 0 vs. 4 +/- 0, P < 0.01), vasculitis (1 +/- 0.7 vs. 2.6 +/- 0.6, P < 0.05), hemorrhage (0.4 +/- 0.6 vs. 3.2 +/- 0.4, P < 0.01), and necrosis (0 +/- 0 vs. 2.4 +/- 0.5, P < 0.005) were significantly reduced in animals treated with CTLA4Ig. Since CTLA4Ig blocks Th1 cell activation in vitro, we compared the levels of Th1 inflammatory cytokines IL-2, gamma-IFN, and TNF-alpha in the two models. The intragraft ratios (CTLA4Ig/control) were IL-2:0.77, gamma-IFN: 1.29, and TNF-alpha:1.33. Thus, CTLA4Ig did not significantly block intragraft production of Th1 cytokines on day 7.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrophobic extracts of a Chinese herb (CMX-13) exhibit potent immunosuppressive properties and prevent acute rejection in a highly histoincompatible model of rat lung transplantation.

BACKGROUND: The potential of higher plants as sources for new immunosuppressive medications is well recognized. In our experiments we investigated the immunosuppressive effect of a highly refined and potent extract of a Chinese herbal preparation, CMX-13, on inhibiting acute allograft rejection (AR) in a highly histoincompatible rat lung transplant model, BN-->LEW, and on lymphocyte activation and cytokine gene expression in vitro. METHODS: Left lung transplants: the control group (group 1) received only dimethylsulfoxide (DMSO) which is the solvent for CMX-13. Group 2 received intramuscular cyclosporin A (CsA, 25 mg/kg) on day 2 posttransplant. Group 3 and 4 received i.p. CMX-13 (0.5 mg/day, low dose and 5 mg/day, high dose, respectively) on day 1, 2, and 3 posttransplant. All animals were killed on day 6 posttransplant. Several pathological categories of inflammation were examined. In vitro experiments: rat spleen cells were incubated with Con A or irradiated stimulator cells with/without serial dilutions of CMX-13 or CsA. Cell proliferation was measured by 3H-thymidine incorporation. mRNA expression of interleukin-2 and interferon-gamma was examined by reverse transcriptase-polymerase chain reaction. RESULTS: The severity of AR in animals receiving high dose CMX-13 was significantly reduced (stage II, P<0.05) compared with controls (stage IV). Significant differences were also seen when more specific parameters of inflammation were examined (necrosis, 0 vs. 1.7+/-1.0, P<0.05; interalveolar hemorrhage, 0 vs. 3.0+/-0.9, P<0.05). The responses seen in the animals treated with high dose CMX-13 were similar to those in the CsA group. CMX-13 inhibited T cell proliferative responses induced by Con A and alloantigen stimulation in a dose-dependent manner that were similar to CsA. Interleukin-2, and interferon-gamma mRNA expression in Con A-stimulated spleen cells was not inhibited by CMX-13 although CsA showed significant inhibition. CONCLUSIONS: 1) CMX-13 significantly reduces the stage of AR and parameters of inflammation in a highly histoincompatible rat lung transplant model. 2) CMX-13 has equal potency to CsA in the inhibition of Con A and alloantigen stimulated rat spleen cell proliferation. 3) CMX-13 showed no inhibitory effects on IL-2 and gamma-IFN mRNA expression, suggesting that its mechanism of action is different from CsA. 4) CMX-13 or derivatives may have potential utility as an immunosuppressive agent(s) in modulation of AR and management of other inflammatory and immunological disorders.

Antithrombin III treatment improves parameters of acute inflammation in a highly histoincompatible model of rat lung allograft rejection.

BACKGROUND: Antithrombin III (AT-III) is an antithrombotic agent with known anti-inflammatory properties that is also known to attenuate acute inflammation, prevent ischemia-reperfusion injury, and disseminated intravascular coagulation (DIC) associated with sepsis and endotoxemia. Here, we examined the ability of AT-III to modify parameters of acute inflammation in a highly histoincompatible model of rat lung allograft rejection (AR). METHODS: After left single lung transplantations (BN-->Lew), recipient animals were treated i.v. with 50 U/kg of human AT-III (low dose group), 500 U/kg of human AT-III (high dose group), or normal saline (control group) on days 2 and 4 posttransplant. All animals were sacrificed on day 6, and several pathological categories of acute inflammation related to AR were scored (0-4). The effect of AT-III on concanavalin A (Con A)-stimulated rat spleen cell proliferation was also examined. RESULTS: The stage of AR, and the degrees of edema, hemorrhage, and necrosis were significantly reduced in the high dose group compared with the control group. AT-III significantly inhibited rat spleen cell proliferation in response to Con A, in a dose-dependent manner. Maximal inhibition was seen at 15 U/ml in culture. Identical inhibition of Con-A-stimulated cultures occurred in both serum free and serum-containing media, indicating that AT-III inhibition of Con-A-stimulated rat spleen cell proliferation is independent of its actions on thrombin. CONCLUSIONS: 1) AT-III treatment significantly improves parameters of acute inflammation seen in a highly histoincompatible model of rat lung AR. 2) AT-III inhibits in vitro T cell proliferation to the potent mitogen Con A, suggesting that protease inhibition may inhibit T cell activation in vitro. 3). The beneficial effects of AT-III on parameters of lung AR relate to the anti-coagulant, anti-inflammatory, and possibly immunoregulatory actions of AT-III.

Subcutaneous leiomyosarcoma with lymphomatous elements: histogenetic implications of an unusual sarcoma.

A 49-year-old woman had an unusual sarcoma that recurred three times in four years. The tumor manifested initially as a subcutaneous abdominal wall nodule composed of lymphohistiocytic malignant lymphoma and spindle cell sarcoma with epithelioid and clear cell elements. The neoplasm recurred first as a spindle cell sarcoma in the lumbar area, second as a malignant lymphoma in the subcutaneous tissue of the right shoulder, and third as a spindle cell sarcoma with lymphohistiocytic areas in the abdominal wall. Ultrastructural studies of this last tumor nodule demonstrated the unequivocal smooth muscle features of the spindle cells. Histogenetic implications of this neoplasm are discussed. It is proposed that the tumor represents an unusual variant of malignant mesenchymoma of soft tissues.

Computerized interactive morphometry in pathology: current instrumentation and methods.

This review is concerned with the current applications of interactive computerized morphometry for diagnostic applications. "Interactive" differs from fully automatic procedures in that the essential function of cell recognition is performed by a trained observer, assisted by a computerized instrument that performs all desirable measurement and processing tasks. This article describes the instrumental needs (video systems, computer, and specialized hardware) and interactive peripherals, in particular, high-resolution touch screens for most common and currently useful video analysis tasks. The authors describe a number of general instrumental procedures with widespread applications (point counting, tracing of contours, graphic standards of size and length, shape analysis, automatic edge detection). Diagnostic procedures are based on the generation of multiple data followed by either multivariate statistical analysis that allows diagnosis of individual cases or hierarchical algorithms. The best current application is the objective study of controversial or difficult cases; there is a possibility that in the future interactive computerized morphometry may become useful for the mass screening of cytologic specimens.

Pulmonary complications of the acquired immunodeficiency syndrome: a clinicopathologic study of 70 cases.

The pulmonary complications of 70 patients with the acquired immunodeficiency syndrome (AIDS) are reviewed. Pneumocystis carinii pneumonia (PCP), present in 67 per cent of the patients, was diagnosed by fiberoptic bronchoscopy with transbronchial biopsies in all of the patients except two adults, who required open lung biopsy, and two children, in whom the infection was detected only at autopsy. Other opportunistic infections, such as cytomegalovirus pneumonitis, mycobacterial infections, invasive candidiasis, toxoplasmosis, cryptococcosis, and histoplasmosis, were more difficult to diagnose by fiberoptic bronchoscopy. In only four cases were these conditions detected during life. Neoplasms and lymphoproliferative processes also presented diagnostic problems, and only one case each of Kaposi's sarcoma and lymphoid interstitial pneumonitis were detected by fiberoptic bronchoscopy. In four other cases these conditions, as well as two pulmonary lymphomas, diffuse large cell immunoblastic type, were detected only at autopsy. Sixty-eight per cent of the patients in this study died, usually with progressive intractable respiratory failure and pulmonary complications that had not been diagnosed during life, including potentially treatable diseases, such as bacterial pneumonias, PCP, nontuberculous mycobacteria, invasive candidiasis, toxoplasmosis, and invasive aspergillosis. The need for earlier detection of pulmonary complications in patients with AIDS is discussed.

The changing spectrum of disease, etiology, and diagnosis of mucormycosis.

During the 20 year interval from 1958 to 1978 a change in the spectrum of disease, etiology, and diagnosis of mucormycosis was observed at The Mount Sinai Hospital. Although the rhinocerebral and pulmonary forms of mucormycosis were still the most frequent forms of disease, hospital acquired cutaneous and subcutaneous infections emerged. Since 1974, 14 of 15 cases of mucormycosis were diagnosed during life. Rizopus species, especially R. rhizopodoformis, have been the etiologic agents identified in 13 of 14 culturally proven cases. The presence or absence of antirhizopus fungistatic activity and antirhizopus antibody in the sera of six of the patients was correlated with the severity of clinical disease. Preliminary results showed a relationship between the extent of disease and the degree of serum fungistatic activity that was independent of antibody production.

Virtual microscopy: high resolution digital photomicrography as a tool for light microscopy simulation.

Recent advances in microcomputers and high resolution digital video cameras provide pathologists the opportunity to combine precision optics with digital imaging technology and develop new educational and research tools. We review recent advances in virtual microscopy and describe techniques for viewing digital images using a microcomputer-based workstation to simulate light microscopic examination, including scanning at low power to select features of interest and zooming to increase magnification. Hardware and software components necessary to acquire digital images of histological and cytological slides, and closely simulate their examination under a light microscope are discussed. The workstation is composed of a MicroLumina digital scanning camera (Leaf Systems, Southborough, MA), light microscope (Olympus Optical Co., Lake Success, NY), Pentium (Intel Corp., Santa Clara, CA) 166 MHz microcomputer configured with 64 megabytes of random access memory (RAM), a MGA Millenium Powerdesk graphics card (Matrox Graphics, Inc., Montreal, Canada) and Photoshop software (Adobe Systems Inc., San Jose, CA) running in a Windows 95 (Microsoft Corp., Redmond, WA) environment. Images with spatial resolutions of up to 2700 x 3400 pixels in 36-bit color, can be displayed simultaneously as distinct images in a montage, or merged into a single composite image file to highlight significant features of a histological or cytological slide. These image files are saved in Joint Photographers Experts Group (JPEG) format using compression ratios of up to 80:1 without detectable visual degradation. The advantages and technical limitations of various workstation components are addressed and applications of this technology for pathology education, proficiency testing, telepathology, and database development are discussed.

Lymphocytic interstitial pneumonia in patients at risk for the acquired immune deficiency syndrome.

Three patients with the acquired immune deficiency syndrome (AIDS) or AIDS-related complex and lymphocytic interstitial pneumonia are reported. All patients presented with progressive dyspnea, nonproductive cough, fever, anorexia, weight loss, and arterial hypoxemia. Chest roentgenograms exhibited bilateral diffuse reticular-nodular densities. The diagnosis of lymphocytic interstitial pneumonia was made by fiberoptic bronchoscopy or open lung biopsy. Two patients were treated with corticosteroids, with significant improvement. The third patient died of pneumonia due to Pneumocystis carinii six months after the diagnosis of lymphocytic interstitial pneumonia was established. Serum antibodies to human immunodeficiency virus (HIV) were demonstrable in the two patients in whom the test was performed. Lymphocytic interstitial pneumonia is probably another pulmonary manifestation of AIDS or AIDS-related complex. Although the clinical presentation may be identical to the more common opportunistic infections, the treatment differs, and the prognosis may be better.

Nontuberculous mycobacteria in pleural fluid. Assessment of clinical significance.

We reviewed the case records of 22 patients from whose pleural fluid a nontuberculous Mycobacterium (NTM) was isolated. Three patients had pleural effusions definitely due to NTM infection, with evidence of NTM infection in other tissues; 16 had pleural effusions of known etiology unrelated to the isolated NTM and no other evidence of NTM infection; and three had pleural effusions of undetermined etiology and no other evidence of NTM infection. The case histories of the three patients with pleural effusions due to NTM are presented and the significance of the isolation of NTM from the remaining 19 patients is discussed. Criteria are proposed for evaluating the significance of NTM isolated from pleural fluid.

Upper airway obstruction secondary to acinic cell carcinoma of the trachea. Use of Nd:YAG laser.

Comprising 1 to 4% of all tumors and 7 to 15% of malignant neoplasms of the major salivary glands, acinic cell carcinoma (ACC) rarely occurs in the respiratory tract. There has been only one case of ACC of the trachea previously reported in the medical literature. A second case of ACC of the trachea associated with upper airway obstruction and its management by Nd: YAG laser and surgical resection is reported.

"Refractoriness" of airflow obstruction associated with isolated lymphocytic bronchiolitis/bronchitis in pulmonary allografts.

The clinical significance of an isolated "lymphocytic bronchiolitis/bronchitis" (grade B) as detected in transbronchoscopic biopsy specimens (TBB) is unclear. We therefore have reviewed the spirometric responses associated with isolated grade B diagnoses and contrasted them with episodes of "acute cellular rejection" (grade A); the latter are manifested by "perivascular lymphocytic infiltration." Because lymphocytic bronchiolitis/ bronchitis is considered a nonspecific histologic pattern that may be observed with either allograft rejection or respiratory infections, episodes were analyzed with respect to the presence (grade B [+] CMV) or absence (grade B [-] CMV) of cytomegalovirus infection. The maximum forced expiratory volume in 1 second (FEV1) during the preceding 3 months was used as a baseline for computing percent change in FEV1 coincident with transbronchoscopic biopsies (delta %FEV1 PRE) and maximum values obtained during the 3 months subsequent to specific therapies (delta %FEV1 POST). All episodes of acute cellular rejection (grades A1 to 4) and symptomatic lymphocytic bronchiolitis/bronchitis (grade B) were treated with "pulsed-dose" methylprednisolone, whereas intravenous ganciclovir was administered to patients at risk for recrudescence of cytomegalovirus. Between March 1, 1989, and September 1, 1995, 366 TBB procedures were performed for clinical indications in 57 lung transplant recipients. Histologic diagnoses with acceptable serial spirometric values included grade A1 (n = 9), grade A2 (n = 27), grade A3 (n = 2), grade B(-)CMV (n = 25) and grade B(+)CMV (n = 9). The delta %FEV1 PRE coincident with TBB were not statistically different for the different histologic groups. For grade A1, delta %FEV1 PRE was -14.6% +/- 5.2% (X +/- SEM); A2, -7.6% +/- 1.8%; B(-)CMV, -14.8% +/- 3.9%; and B(+)CMV, -14.8% +/- 2.3%. After treatment, the delta %FEV1 POST, relative to baseline values, were for grade A1, -8.8% +/- 7.1%, A2, +0.26% +/- 2.6%; B(-)CMV, -12.0% +/- 3.8%; and B(+)CMV, -6.2% +/- 2.8%. The delta %FEV1 POST values after pulsed methylprednisolone were significantly greater for histologic grade A2 than grade B(-)CMV (unpaired Student's t test, P < 0.01; 95% confidence interval for the difference of means: 3.34% to 21.2%). Grade A2 rejection was associated with spirometric improvement to within 10% of baseline values in 52% of episodes; whereas with grade B(-)CMV, this salutary response was observed in only 32% of episodes. Bronchiolitis obliterans syndrome stage 1b developed in 13 of 20 (65%) recipients, approximately 7.9 +/- 3.4 months after detection of histologic grade B and 21.2 +/- 9.5 months after transplantation. We conclude that the relative "refractoriness" of histologic grade B most likely reflects a continuum of bronchiolitis obliterans after lung transplantation and, hence, may warrant different immunosuppressive strategies. Furthermore, spirometric decrement associated with acute cellular rejection (grade A) may be ameliorated, but often not completely reversed, after pulsed methylprednisolone. We speculate that surveillance TBB may prove rewarding by enabling an earlier detection of these histologic diagnoses before the development of physiologic impairment.