Early mechanisms of neutrophil activation and transmigration in acute lung injury.

Introduction:Neutrophil transmigration is multifactorial and primarily driven by selectins and ß2-integrins (CD11b/CD18), whose expression are dependent on the underlying stimulus. Ventilator-induced lung injury (VILI) results in a predominantly CD18-independent mechanism of neutrophil recruitment, while direct endotoxin-induced lung injury results from a CD18-dependent mechanism. We previously observed that lack of NADPH oxidases DUOX1 and DUOX2 resulted in reduced neutrophil influx in a VILI model of lung injury but had no influence on neutrophil influx after LPS exposure. Based on these observations, we hypothesized that DUOX1/DUOX2 are an important component of CD18-independent mechanisms of neutrophil recruitment in the lung. Methods:We exposed Duoxa -/- (KO) mice and Duoxa +/+ (WT) mice to either an intratracheal exposure of lipopolysaccharide (LPS/endotoxin)-or high tidal volume ventilation and compared expression of neutrophil markers between groups. WT mice (129S6/SvEvTac) were obtained from Taconic Biosciences (One Discovery Drive Suite 304; Rensselaer, NY 1244) and were allowed to acclimatize for one week prior to study enrollment. KO mice were generated as previously described [Grasberger 2012] and bred in-house on a 129S6 background. We provided positive-pressure ventilation at a tidal volume of 10 ml/kg with 2 cmH20 positive end-expiratory pressure (PEEP). Mice were assigned to groups consisting of KO (n = 5) and WT (n = 5) in each group and divided into non-ventilated, positive-pressure ventilation, or LPS IT exposure groups. Positive-pressure ventilation was instituted for 4-h using a FlexiVent (Flexiware 8.1, Scireq, Montreal, QC, Canada). Lipopolysaccharide (Salmonella enterica serotype tryphimurium L6143, Millipore Sigma) was administered via an intratracheal (IT) route at a dose of 0.1 mg/kg. Mice were humanely euthanized at 4-h post-injection consistent with the UC Davis IAUCAC-approved protocol. Results:As previously observed, neutrophilic influx into the airways was significantly impaired in the Duoxa -/- (KO) mice after VILI, but not after LPS exposure. LPS-induced lung injury resulted in upregulation of CD11b+ neutrophils and shedding of CD62L and CD162 regardless of DUOX expression, whereas VILI resulted in upregulation of CD49+ neutrophils in the Duoxa +/+ (WT) mice but not the Duoxa -/- (KO) mice. Conclusion:Our data suggest DUOX is required for CD18-independent mechanisms of neutrophil recruitment in the lung induced by acute lung injury, but not for canonical CD18depedent mechanisms after LPS exposure.

Diagnostic yield of cytologic analysis of pericardial effusion in dogs.

BACKGROUND: Pericardial effusion cytology is believed by many to be of limited value, yet few studies have evaluated its diagnostic utility. OBJECTIVES: To determine the diagnostic utility of cytologic analysis of pericardial effusion in dogs and to determine if consideration of additional data could improve the diagnostic yield. ANIMALS: Two hundred and fifty-nine dogs with cytologic analysis of pericardial effusion performed between April 1990 and June 2012. METHODS: Electronic medical records from a university teaching hospital were retrospectively reviewed; signalment, complete blood count, serum biochemistry, cytologic analysis of pericardial effusion, and echocardiographic data were recorded. Cytology was classified as diagnostic (infectious or neoplastic) or nondiagnostic (hemorrhagic or other) and groups were compared with multiple Student's t-tests. RESULTS: Cytology was grouped as nondiagnostic (92.3%) or diagnostic (7.7%) and characterized as hemorrhagic (90%), neoplastic (4.6%), infectious (3.1%), or other (2.3%). Overall cytologic analysis of pericardial effusion diagnostic utility was 7.7% and increased to 20.3% if the effusion hematocrit (HCT) <10%; echocardiographic evidence of a mass did not result in a significant increase in the diagnostic utility. CONCLUSIONS AND CLINICAL IMPORTANCE: The diagnostic utility of cytologic analysis of canine pericardial effusion is variable depending on the underlying etiology. In this group of dogs, the diagnostic yield of cytologic analysis was greater for pericardial effusion samples in which the HCT was less than 10%.

Prevalence of donor-specific anti-HLA antibodies during episodes of renal allograft rejection.

BACKGROUND: Recent studies suggest that the appearance of anti-HLA antibodies in the early posttransplant period is associated with an increased incidence of acute and chronic rejection months later. However, very little is known about the prevalence of anti-HLA antibodies at the time that the rejection episodes are diagnosed. The purpose of this study was to analyze retrospectively 420 sera from 263 renal allograft recipients who were readmitted to the hospital for any reason between 1989 and 1998 in order to determine if a correlation existed between the presence of donor-specific anti-HLA antibodies and graft rejection. METHODS: Sera were assayed for IgG HLA class I and II antibodies by ELISA. The ELISA results were analyzed using contingency tables with Fisher's exact test and compared with mismatched antigens in the donor. RESULTS: Antibodies to donor HLA class I molecules in the posttransplant sera were extremely rare, occurring in only 6 of the 420 sera (1.4%) analyzed. Antibodies to donor class II antigens were slightly more common, occurring in 25 of the 420 sera (6%). In 21 of these 25 cases (84%), the presence of donor-specific HLA class II antibodies was associated with episodes of either acute (n=14) or chronic rejection (n=7). Five patients had antibodies to both class I and class II donor antigens, and all five of them lost their grafts to rejection. CONCLUSION: Although the presence of donor-specific HLA antibodies presented a significant risk for acute or chronic rejection, 77% of all acute and chronic rejections occurred in patients without detectable HLA antibodies.

Diphtheria toxin A gene-mediated HIV-1 protection of cord blood-derived T cells in the SCID-hu mouse model.

The reconstitutive potential of CD34+-derived cord blood (CB) cells, transduced with a regulated diphtheria toxin A (DT-A) chain gene, was examined in SCID-hu mice harboring a conjoint organ composed of human thymus and liver (thy/liv). The DT-A-transduced cells, injected directly into the thy/liv organ, showed the same engraftment potential as control CB cells transduced with the non-DT-A parental vector. CB cells, distinguishable from the thy/liv cells by the HLA marker B7, were preferentially maintained in ex vivo culture. In the thy/liv organ, the engrafted CB cells represented >80% of the total cells. A majority of cells (>70%) in the thy/liv organ were also CD4+CD8+, as would be expected of maturing thymocytes. The incidence of double-positive cells was highest at 44 days (compared with 30 days and 80 days) after injection of CB cells. This suggested that a minimum time was required to achieve optimal proliferation of cells in the thy/liv organ but that, at later times, all of the early cells had matured. Thus, the population used for engraftment contained early cells but not self-renewing cells. The double-positive cells matured rapidly into single-positive cells (either CD4+ or CD8+) when placed in ex vivo culture. Marked cells (neo+) could readily be detected in the thy/liv-derived cells. The cells transduced with DT-A showed long-term protection in ex vivo culture against HIV T lymphotropic isolate NL4-3. This study shows that DT-A-transduced cells had no apparent disadvantage in engraftment of the thy/liv organ and did not have any toxic effects in vivo. Such cells were protected against HIV infection even when challenged more than 2 months after transduction and after a 44-day engraftment period in the thy/liv mice. These data support the feasibility of toxin gene therapy as a strategy for HIV infection.

Video endoscopic transanal-rectal tumor excision.

BACKGROUND: Transanal resection of benign and selected malignant rectal tumors is a well accepted surgical technique. The use of a stereoscopic microsurgical technique, as originally described by Buess et al in 1984, has been shown to improve the results of standard transanal resection by allowing precise, full thickness resections up to 24 cm from the anal verge. Transanal endoscopic microsurgery (TEM) has failed to gain widespread popularity for two reasons: The proprietary instrument set is expensive and complex ($68,000 and 30 components), and the procedure is difficult to master technically. We present our results with a modification of the TEM instrument that incorporates a standard laparoscope and video camera as well as standard laparoscopic instruments. METHODS: Four surgeons have been trained to date. Details of the training curriculum are presented. The technique of videoendoscopic transanal tumor resection (VTEM) is described. A prospective data base was maintained of all VTEM cases. This was reviewed for this study to determine indications, operative times, complications and outcomes. RESULTS: Four surgeons performed 27 VTEM cases between August 1994 and June 1996. The average age was 69 years and the majority (16) of patients were ASA III. Pre-op diagnosis was benign polyp in 25 patients and adenocarcinoma in 2. Average operating time was 127 minutes (49 to 280 minutes), and was longer during a surgeon's first 5 cases and for lesions more than 16 cm from the anal verge. Operative problems were rare (4%) and post-op complications (incontinence 2, late bleeding 1, adenoma recurrence 1) were seen in 15%. CONCLUSIONS: VTEM can be taught successfully to GI and colorectal surgeons using a format similar to that used for advanced laparoscopic courses. The use of already available laparoscopes and instruments decreases the initial costs of the set-up. Results are good, with low rates of complications and recurrence and a very short hospital stay. The patient benefits from an effective, minimally invasive alternative to open surgery.

A rapid flow cytometry assay for HLA antibody detection using a pooled cell panel covering 14 serological crossreacting groups.

Many studies have demonstrated the usefulness of flow cytometry crossmatching (FC-XM) for selection of regraft recipients, and more recently this assay has been shown to correlate with allograft survival in primary cadaveric transplant patients. The need now exists for a practical antibody screening procedure which uses the same methodology. We describe here a simple and sensitive method to screen for HLA antibodies by FC using a pool of 6 cells selected to cover the 14 serological crossreacting groups defined by Rodey. Screenings of 367 sera (255 primary transplant sera, 112 regraft sera) received for monthly antibody testing were performed by both pooled cell FC and complement-dependent cytotoxicity (CDC) assays. Forty of these sera were also FC-screened using a panel of 16 individual cells for comparison with the pooled cell FC screenings. Analysis indicated a strong correlation between the pooled FC-PRA and the individual cell panel FC-PRA (P = .0001) with mean values of 60% and 73%, respectively. Only 2 of the 40 sera screened by both FC methods resulted in PRAs that differed by > 40%. The majority (82%) of the primary patients did not exhibit HLA antibodies by CDC--however, 22% of the CDC negative patients were positive by flow cytometry. Females were more likely to be positive by FC (35%) than males (16%) (P = .0001). Similarly, black patients were more likely to have FC-demonstrable antibodies (28%) than white candidates (14%) (P = .014). The regraft patients who tested positive by either or all methods had a mean PRA for CDC, pooled FC-PRA, and individual cell FC-PRA of 40, 75, and 85, respectively. FC-PRA proved to be a more sensitive technique in both primary and regraft patients.

Technical details of intraoperative lymphatic mapping for early stage melanoma.

The initial route of metastases in most patients with melanoma is via the lymphatics to the regional nodes. However, routine lymphadenectomy for patients with clinical stage I melanoma remains controversial because most of these patients do not have nodal metastases, are unlikely to benefit from the operation, and may suffer troublesome postoperative edema of the limbs. A new procedure was developed using vital dyes that permits intraoperative identification of the sentinel lymph node, the lymph node nearest the site of the primary melanoma, on the direct drainage pathway. The most likely site of early metastases, the sentinel node can be removed for immediate intraoperative study to identify clinically occult melanoma cells. We successfully identified the sentinel node(s) in 194 of 237 lymphatic basins and detected metastases in 40 specimens (21%) on examination of routine hematoxylin-eosin-stained slides (12%) or exclusively in immunohistochemically stained preparations (9%). Metastases were present in 47 (18%) of 259 sentinel nodes, while nonsentinel nodes were the sole site of metastasis in only two of 3079 nodes from 194 lymphadenectomy specimens that had an identifiable sentinel node, a false-negative rate of less than 1%. Thus, this technique identifies, with a high degree of accuracy, patients with early stage melanoma who have nodal metastases and are likely to benefit from radical lymphadenectomy.

Lymphatic drainage of skin to a sentinel lymph node in a feline model.

To determine the feasibility of selective lymphadenectomy, the authors developed a feline model to identify and determine the utility of mapping dyes for this purpose. Adult cats were injected intradermally with a variety of mapping substances to determine whether the anatomic site of injection had a predictable pattern of drainage to a particular lymph node. Isosulfan blue provided the optimal mapping material. Injection of isosulfan blue intradermally into the skin of the medial thigh consistently led to coloration of the central lymph node, whereas intradermal abdominal wall injections and intradermal lateral thigh injections resulted in coloration of the lateral lymph node. Intradermal injections into skin about the perineum resulted in coloration of the most medial lymph node only. The feline model proved to a useful model to examine the utility of mapping dyes and to demonstrate dermal lymphatics. The predictable pattern of drainage of the skin in this feline model supports the feasibility of selective lymphadenectomy.

Isolated subclavian artery dissection after blunt trauma.

We present a case report of a patient with an isolated dissection of the subclavian artery after blunt trauma. The patient who was admitted to our center after a motor vehicle accident, complained of chest and neck pain and physical findings of diminished left extremity pulses. Arteriography showed an occluded subclavian artery with the possibility of a dissection. The dissection was confirmed at surgery with the proximal extent originating just distal to the origin of the vertebral artery. The distal extent of the dissection was not determined. Operative repair was performed by a carotid-to-subclavian artery bypass obliterating the false lumen of the dissection with a running vascular anastomosis. The patient, who was discharged 5 days after repair, had normal extremity neurovascular function at 4 months follow-up.

Natural history and selective management of in transit melanoma.

Ninety five patients who developed in transit melanoma either as their initial site of recurrent melanoma or following regional node metastases were retrospectively reviewed. In transit melanoma occurred most frequently on the lower extremity and appeared to be associated with deeply invasive primary tumors. The median time to development of in transit melanoma was 16 months. Eighty-two (86%) of these patients have progressed to systemic disease from 2 to 244 months (median 16 months) following the development of in transit melanoma, and 72 (79%) died (median survival 19 months). Survival appears to correlate with the extent of in transit melanoma and with the disease-free interval. These findings suggest that in transit melanoma represents an early manifestation of systemic disease, warranting careful clinical follow-up and perhaps systemic treatment, when effective therapy becomes available. However, some patients will respond to local immunotherapy, surgical excision, and isolated limb perfusion and will enjoy significant length and quality of life. This sequential approach remains the treatment of choice for this manifestation of metastatic melanoma.

Detection of malignant melanoma with iodine-123 iodoamphetamine.

Iodoamphetamine (IMP) was shown by in vitro assay to have a high uptake by human melanotic melanoma cells, as compared to amelanotic melanoma cells. Eleven patients with proven malignant melanoma (MM) and 3 normal subjects were imaged at 2-4 hr and 16-24 hr after the i.v. injection 5 mCi (185 MBq) of [123I]IMP. One patient had a recurrent tumor that was subsequently shown to be squamous cell carcinoma. The index lesion was not visualized in the three patients with amelanotic melanomas. The index lesion/lesions were visualized in six of the seven other patients, except for 4/16 nodules in one patient. The seventh patient had a large, necrotic melanotic tumor that was not visualized, but an unsuspected lesion in the iliac nodes was detected. Multiple unsuspected lesions were detected in a second patient. While many lesions were seen at 2-4 hr, all lesions (other than a patient with small bowel disease) were seen best at 16-24 hr. No eye uptake was observed in any patient or control subject. Testicular uptake was seen in all males at 16-24 hr. Iodine-123 IMP appears to be a useful agent for the detection and follow-up of patients with melanotic MM.

Surgical treatment of lymph nodes with metastatic melanoma from unknown primary site.

To determine the prognosis of patients with lymph node metastases from an unknown primary melanoma, we retrospectively reviewed the clinicopathologic features of 188 such patients treated from 1971 through 1986 and compared their records with those of patients with clinical stage II melanoma with known primary lesions. Patients with lymph node metastases from an unknown primary melanoma represented 4.6% of all patients with melanoma treated during that period. The five- and ten-year survival rates were 42% and 40%, respectively (median, 37 months). When stratified by number of tumor-containing lymph nodes, there was no significant difference in survival between patients with an unknown primary melanoma and lymph node metastases and those with clinical stage II melanoma and known primary sites. The prognosis of the former patients is no worse than that of patients with lymph node metastases from a known primary site and should be treated in a comparable manner.

Natural history of surgically treated mucosal melanoma.

Thirty-three patients with melanoma arising in a mucosal site were reviewed. Sixteen patients were treated either with abdominoperineal resection or radical vulvectomy and superficial inguinal lymphadenectomy. Three patients were treated palliatively. Fourteen patients were treated conservatively with local excision, wide local excision, and radiation therapy. One patient received systemic chemotherapy and radiation therapy. Local recurrence developed in seven patients. The overall survival rate was poor. Neither local control nor survival appeared to be influenced by the initial surgical approach.

Histologic features relating to prognosis in synovial sarcoma.

Although the American Joint Commission has classified all synovial sarcomas as "high grade," histologic subtypes can be identified. By histologically subclassifying synovial sarcoma tumors according to percent glandularity and mitotic rates, the authors were able to define high-risk and low-risk patients. Charts and original pathologic slides were reviewed on 45 synovial sarcoma patients. With a 41-month median follow-up, the low-risk patients showed 100% survival, whereas the high-risk patients showed 37% survival.

The validity of assessing mental health needs with social indicators.

Selection of the appropriate number and types of social indicators for use in mental health planning has been a perennial problem. Social indicators have been associated with several concepts (quality of life, community disorganization, populations at risk) of varying relevance for planning mental health services, and abstracting social indicators from these conceptual domains poses a variety of validity issues. The issues are discussed, and the viability of social indicators in mental health planning is reexamined.