Marked differences in local bone remodelling in response to different marrow stimulation techniques in a large animal.

Marrow stimulation, including subchondral drilling and microfracture, is the most commonly performed cartilage repair strategy, whereby the subchondral bone plate is perforated to release marrow-derived cells into a cartilage defect to initiate repair. Novel scaffolds and therapeutics are being designed to enhance and extend the positive short-term outcomes of this marrow stimulation. However, the translation of these newer treatments is hindered by bony abnormalities, including bone resorption, intralesional osteophytes, and bone cysts, that can arise after marrow stimulation. In this study, three different marrow stimulation approaches - microfracture, subchondral drilling and needle-puncture - were evaluated in a translationally relevant large-animal model, the Yucatan minipig. The objective of the study was to determine which method of marrow access (malleted awl, drilled Kirschner wire or spring-loaded needle) best preserved the underlying subchondral bone. Fluorochrome labels were injected at the time of surgery and 2 weeks post-surgery to capture bone remodelling over the first 4 weeks. Comprehensive outcome measures included cartilage indentation testing, histological grading, microcomputed tomography and fluorochrome imaging. Findings indicated that needle-puncture devices best preserved the underlying subchondral bone relative to other marrow access approaches. This may relate to the degree of bony compaction occurring with marrow access, as the Kirschner wire approach, which consolidated bone the most, induced the most significant bone damage with marrow stimulation. This study provided basic scientific evidence in support of updated marrow stimulation techniques for preclinical and clinical practice.

Neutrophils alter epithelial response to Porphyromonas gingivalis in a gingival crevice model.

A gingival crevice model (epithelial cell-Porphyromonas gingivalis-neutrophil) was established and used to profile gingipain, matrix metalloproteinase (MMP), MMP mediators [neutrophil gelatinase-associated lipocalin (NGAL) and tissue inhibitor of metalloproteinases 1 (TIMP-1)] and cytokine networks. Smoking is the primary environmental risk factor for periodontitis. Therefore, the influence of cigarette smoke extract (CSE) was also monitored in the same model. Porphyromonas gingivalis alone induced low levels of interleukin-1ß and interleukin-8 from epithelial cells, but high levels of both cytokines were produced on the addition of neutrophils. Exposure to CSE (100 and 1000 ng ml(-1) nicotine equivalency) significantly compromised P. gingivalis-induced cytokine secretion (both P < 0.05). P. gingivalis induced impressive secretion of NGAL (P < 0.05) that was not influenced by CSE. The influence of CSE on gingipain production was strain-specific. Purified gingipains effectively and rapidly degraded both TIMP-1 and MMP-9. Induction of large amounts of NGAL, degradation of TIMP-1, and increased gingipain activity would each be expected to prolong collagen degradation and promote disease progression. However, gingipains also degrade MMP-9. Hence, P. gingivalis exerts a complex influence on the proteolytic balance of a gingival crevice model. Exposure to CSE reduces the proinflammatory cytokine burden, which may be expected to promote P. gingivalis survival. In addition to novel findings that provide mechanistic insight into periodontal disease progression, these results are in keeping with the recognized clinical dogma of decreased inflammation/increased disease in smokers. This straightforward gingival crevice model is established as a suitable vehicle for the elucidation of mechanisms that contribute to susceptibility to periodontitis.

Failures, re-operations, and complications after autologous chondrocyte implantation--a systematic review.

OBJECTIVE: To determine and compare failure, re-operation, and complication rates of all generations and techniques of autologous chondrocyte implantation (ACI). METHODS: A systematic review of multiple medical databases was performed according to PRISMA guidelines. Levels I-IV evidence were included. Generations of ACI and complications after ACI were explicitly defined. All subject and defect demographic data were analyzed. Modified Coleman Methodology Scores (MCMSs) were calculated for all studies. RESULTS: 82 studies were identified for inclusion (5276 subjects were analyzed; 6080 defects). Ninety percent of the studies in this review were rated poor according to the MCMS. There were 305 failures overall (5.8% subjects; mean time to failure 22 months). Failure rate was highest with periosteal ACI (PACI). Failure rates after PACI, collagen-membrane cover ACI (CACI), second generation, and all-arthroscopic, second-generation ACI were 7.7%, 1.5%, 3.3%, and 0.83%, respectively. The failure rate of arthrotomy-based ACI was 6.1% vs 0.83% for all-arthroscopic ACI. Overall rate of re-operation was 33%. Re-operation rate after PACI, CACI, and second-generation ACI was 36%, 40%, and 18%, respectively. However, upon exclusion of planned second-look arthroscopy, re-operation rate was highest after PACI. Unplanned re-operation rates after PACI, CACI, second-generation, and all-arthroscopic second-generation ACI were 27%, 5%, 5%, and 1.4%, respectively. Low numbers of patients undergoing third-generation ACI precluded comparative analysis of this group. CONCLUSIONS: Failure rate after all ACI generations is low (1.5-7.7%). Failure rate is highest with PACI, and lower with CACI and second-generation techniques. One out of three ACI patients underwent a re-operation. Unplanned re-operations are seen most often following PACI. Hypertrophy and delamination is most commonly seen after PACI. Arthrofibrosis is most commonly seen after arthrotomy-based ACI. Use of a collagen-membrane cover, second-generation techniques, and all-arthroscopic, second-generation approaches have reduced the failure, complication, and re-operation rate after ACI.

Mullerian inhibiting substance suppresses tumor growth in the C3(1)T antigen transgenic mouse mammary carcinoma model.

Mullerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro. To extend the use of MIS to treat breast cancer, it is essential to test the responsiveness of mammary tumor growth to MIS in vivo. Mammary tumors arising in the C3(1) T antigen mouse model expressed the MIS type II receptor, and MIS in vitro inhibited the growth of cells derived from tumors. Administration of MIS to mice was associated with a lower number of palpable mammary tumors compared with vehicle-treated mice (P=0.048), and the mean mammary tumor weight in the MIS-treated group was significantly lower compared with the control group (P=0.029). Analysis of proliferating cell nuclear antigen (PCNA) expression and caspase-3 cleavage in tumors revealed that exposure to MIS was associated with decreased proliferation and increased apoptosis, respectively, and was not caused by a decline in T antigen expression. The effect of MIS on tumor growth was also evaluated on xenografted human breast cancer cell line MDA-MB-468, which is estrogen receptor- and retinoblastoma-negative and expresses mutant p53, and thus complements the C3(1)Tag mouse mammary tumors that do not express estrogen receptor and have functional inactivation of retinoblastoma and p53. In agreement with results observed in the transgenic mice, MIS decreased the rate of MDA-MB-468 tumor growth and the gain in mean tumor volume in severe combined immunodeficient mice compared with vehicle-treated controls (P=0.004). These results suggest that MIS can suppress the growth of mammary tumors in vivo.

Enzyme immunoassays for antinuclear antibodies.

To evaluate the potential advantages and disadvantages of the EANA, the immunopathology laboratory of the Henry Ford Health System compared the qualitative and quantitative results from several EANA assays to those from a well-standardized FANA assay and then correlated them with the clinical data.

IgG anticardiolipin antibody titer > 40 GPL and the risk of subsequent thrombo-occlusive events and death. A prospective cohort study.

BACKGROUND: Anticardiolipin antibodies (aCL) have been associated with an increased risk of stroke and thrombo-occlusive events. Little is known about the influence of aCL on recurrent thrombo-occlusive events. METHODS: Consecutively identified patients (n = 132) with focal cerebral ischemia [stroke = 112, transient ischemic attack (TIA) = 20] harboring aCL of at least 10 GPL units at the time of their index event were prospectively followed to estimate the effect of aCL titer on time to and risk of subsequent thrombo-occlusive events (stroke, TIA, deep venous thrombosis, pulmonary embolism, myocardial infarction) and death. On the basis of prior literature, we divided patients into those with aCL < or = 40 GPL (n = 111; mean age, 63 +/- 14 years; mean follow-up, 1.95 years) and those with aCL > 40 GPL (n = 21; mean age, 54 +/- 20 years; mean follow-up, 1.50 years). RESULTS: There was no difference between groups for prevalence of hypertension, diabetes mellitus, cigarette smoking, atrial fibrillation, prior TIA, or sex. The GPL > 40 group was younger (54 +/- 20 versus 63 +/- 14 years; P = .055), had more prior strokes [9/21 (48%) versus 27/111 (20%); P = .030], more frequent subsequent thrombo-occlusive events and death [15/21 (71%) versus 51/111 (48%); P = .030], and a shorter median time (years) to event (0.15 versus 0.61, log rank P = .005). The risk ratio for recurrent event and death with GPL > 40 obtained from Cox proportional hazards models, adjusted for prior strokes, prior TIAs, hypertension, diabetes mellitus, atrial fibrillation, and cigarette smoking was 1.9 (95% confidence interval, 1.0 to 3.5; P = .051). CONCLUSIONS: Our data suggest that subsequent thrombo-occlusive events and death after focal cerebral ischemia associated with IgG aCL may occur sooner and more frequently with GPL > 40.

An algorithmic approach to thyroid function testing in a managed care setting. 3-year experience.

An algorithm (directed thyrotropin [TSH], directed thyroid testing algorithm [DRTSH]) for the initial evaluation and monitoring of thyroid function was established in our institution in 1990. The algorithm begins with measurement of TSH by a sensitive immunoassay. If TSH is either < 0.4 mU/L or > 5.5 mU/L, a free thyroid index (T4 x Resin Uptake Ratio (RU)) is automatically performed on the same sample on the same day. In the setting of a large, predominately outpatient, prepaid health care population, the algorithm reduces unnecessary testing and focuses resources on the patients who need it. Three years after its introduction, physician acceptance of this approach is high ( > 90%), test utilization is reduced, test turnaround time is reduced, and significant cost-savings can be demonstrated.

Idiopathic CD4+ T-lymphocytopenia: analysis of a patient with selective IgA deficiency and no evidence of HIV infection.

Idiopathic CD4+ T-lymphocytopenia (ICL) in HIV-seronegative patients is a newly described, rare entity. The common underlying abnormality is a usually stable depletion in CD4+ lymphocytes in patients, some of which have unexplained opportunistic infections. We present a previously unreported condition of an asymptomatic individual with CD4+ T-lymphocytopenia and a selective IgA deficiency. The subject is a 35-year-old healthy white male with a documented 5-year history of low CD4+ T cell counts. He has been repeatedly HIV seronegative and has no risk factors for HIV infection. Data were obtained from several laboratories over a 5-year period and include standard WBC differentials, HIV testing, serum immunoglobulin quantitation, mitogen stimulation assays, diphtheria and tetanus antitoxin titers, and flow cytometric immunophenotyping. The composite results show a subject with a normal white blood cell count, an absolute lymphopenia, a slight granulocytosis, and a selective IgA deficiency. Leukocyte subset analyses show essentially normal B but significantly altered T cell phenotypes. The normal CD4:CD8 ratio shows extreme inversion, primarily due to CD4 T-lymphocytopenia.

Adhesive glycoproteins CD11a and CD18 are upregulated in the leukocytes from patients with ischemic stroke and transient ischemic attacks.

Leukocytes may have an important role in the pathogenesis of brain injury after ischemia. Expression of adhesion molecules on leukocytes and/or endothelia is needed for leukocytes to adhere to endothelia and infiltrate into the injured brain. The purpose of the present pilot study is to delineate whether the expression of leukocyte adhesion molecules, CD11a and CD18, are upregulated in patients with ischemic stroke and transient ischemic attack. Ten patients with ischemic stroke, 6 with transient ischemic attack (TIA), and 11 age and risk factor matched controls were studied. Using immunofluorescence phenotyping and flow cytometry, leukocyte membrane expression of CD11a and CD18 were measured within 72 h after onset of ischemia. Follow-up measurements were performed at 5-7 days after ictus in 6 patients with stroke, and at 3-5 days after ictus in 3 patients with TIA. CD11a immunofluorescence (IF) was significantly increased within 72 h after onset of symptoms in patients with stroke as well as TIA compared with the control group (p < 0.017). IF of CD18 also increased in both patient groups, but significance was reached only in the TIA group (p < 0.05). No difference of CD11a and CD18 IF was detected between stroke and TIA groups. Follow-up measurement of CD11a and CD18 showed a trend of decrease, but CD11a IF remained significantly elevated compared with the control group (p < 0.017). Expression of leukocyte adhesion molecules CD11a, and CD18 are upregulated in patients with ischemic stroke and TIA. Although these data are preliminary, our data suggest that these molecules are associated with cerebrovascular disorders including ischemic stroke and TIA.

Peripheral T-cell non-Hodgkin's lymphoma following treatment of nodular lymphocyte predominance Hodgkin's disease.

Previous reports have suggested that nodular lymphocyte predominance Hodgkin's disease (NLPHD) is a germinal center-derived B-cell lymphoma that is distinct from other types of Hodgkin's Disease. A relationship between NLPHD and simultaneous or subsequent development of large-cell (LC) non-Hodgkin's lymphoma (NHL) has been established. Both Reed-Sternberg cell variants in NLPHD and NHL cells in these cases express B-cell-associated antigens, and in some cases the B-cell lineage of the NHL has been confirmed by immunoglobulin gene rearrangement studies. The B-cell phenotype and the indolent course of both lymphomas suggest histologic progression of NLPHD to B-cell NHL, rather than a de novo LCNHL unrelated to Hodgkin's Disease. We report a unique case of T-large-cell lymphoma (TLCL) following successful chemotherapy of NLPHD. A 54-year-old male was treated with seven cycles of mechlorethamine, vincristine, procarbazine, prednisone chemotherapy for NLPHD and 4 years later developed recurrent adenopathy. Lymph node biopsy showed a diffuse LCNHL. Frozen section immunotyping and gene rearrangement studies confirmed the diagnosis of TLCL. To our knowledge, this case represents only the second report of TLCL associated with NLPHD and is of significance in that: (1) it demonstrates that T-cell neoplasia can occur in the setting of NLPHD; (2) this case does not appear to represent histologic progression of NLPHD and most likely represents de novo disease that may be secondary to chemotherapy; and (3) the clinical course may differ from the favorable prognosis seen in NLPHD associated with B-cell NHL.

The adversarial alliance: developing therapeutic relationships between families and the team in brain injury rehabilitation.

Families of individuals who have survived brain injuries experience significant distress, and may resist accepting their relative's neurobehavioural deficits. Staff who work with brain-injured patients and their relatives are charged with the seemingly paradoxical task of helping families support rehabilitative efforts and be goal-oriented, while simultaneously communicating often negative realities about prognosis. In the midst of what may be an intermittently conflict-laden relationship, families and staff must become synergistically involved in a therapeutic partnership. This paper defines aspects of this 'adversarial alliance' which is often established between families and staff. The relationship between patient discharge outcome and perceived family stress and satisfaction with the rehabilitation programme was reviewed. Data analyses yielded the following conclusions: families evaluated retrospectively to have been 'highly stressed' were also perceived to experience more conflict with the rehabilitation team; family stress was related to poorer adjustment to the patient's disability (at admission); greater family/team conflict correlated with lower cognitive and physical functioning at admission, longer length of stay, younger patient age, and lower programme satisfaction. Implications for programme development and treatment guidelines are discussed.

Megakaryoblastic termination of myeloproliferative disorders.

Megakaryoblastic termination of myeloproliferative disorders is rare. The morphology of megakaryoblastic transformation can be subtle and is often mistaken for myeloid or lymphoid proliferations. Previously reported observations suggest a relatively poor prognosis for this category of patients, making precise diagnosis imperative. A multifaceted approach using morphology, ultrastructure, cytochemistry, and immunological membrane analysis may be helpful. We present two cases of myeloproliferative disorder with aggressive megakaryoblastic phases (myelofibrosis with agnogenic myeloid metaplasia and chronic myeloid leukemia with blast crisis). The clinical course is described and the results of the morphological, cytochemical, ultrastructural, and cytogenetic studies of both cases are presented. In addition, immunochemical studies (flow cytometry) and platelet function studies (aggregation, beta-thromboglobulin, and platelet factor IV release) were done for one of these patients.

Quality control in flow cytometry for diagnostic pathology. I. Cell surface phenotyping and general laboratory procedures.

Flow cytometric analyses have become commonplace in the clinical laboratory for determining cell lineage and for quantitation of cells bearing a given phenotype. Because these assays are being conducted to support diagnoses or assist in determining therapy, it is crucial to ensure that these tests are highly accurate and reproducible within a laboratory and among laboratories involved in similar endeavors. This quality assurance has been slow evolving in clinical flow cytometry for a variety of reasons: the exquisite sensitivity and delicacy of the instrumentation that recognize previously undetectable variations in staining; the constant improvement of the hardware and software; the rapid development of new techniques and reagents of clinical interest; and the failure of any existing specialty or subspecialty to encompass all aspects of flow cytometry. This article provides an overview of quality assurance necessary for the flow cytometric analysis of cell surface markers. Practical experience, published studies, and suggested guidelines from accreditation agencies have been combined to develop the text.

Recent advances in flow cytometric techniques for cancer detection and prognosis.

The application of flow cytometry to cancer diagnosis and the prediction of tumor behavior continues to expand. The ability of flow cytometry to examine multiple parameters of large numbers of individual cells is being exploited increasingly to characterize neoplastic processes better. This will allow closer examination of tumor heterogeneity and identification of subpopulations with different behavioral patterns. Flow cytometry is being used with greater frequency in attempts to predict tumor behavior and response to therapy. Flow cytometry may have the most to offer in this area. Diagnosis of cancer by routine histopathological examination will not be replaced by flow cytometry; flow cytometry will be used in conjunction with morphological descriptions. The detection of proliferation antigens and oncogene products, as well as cell cycling, provides information on neoplastic progression that is not readily obtainable by other methods. In addition, flow cytometry will undoubtedly be used to measure other features of neoplastic cells, such as enzyme levels and ion fluxes, which may better characterize the behavior of the tumor. Advances in flow cytometry instrumentation, light sources, fluorochrome development, and basic aspects of cellular and molecular biology will continue to permit this technology to define neoplastic cells and their behavior better, resulting in both improved patient care and a better understanding of tumor biology.

Detection of the ras p21 gene product in human leukemias by flow cytometry.

To date, the detection of oncogene products in human neoplasms has relied primarily upon immunoblot analysis of specimen homogenates. Herein are reported the results of a study using flow cytometry to evaluate the expression of the ras p21 gene product in both fresh and cryopreserved specimens of human acute leukemias. Cell lines known to express ras p21 were used as positive controls and normal peripheral blood was used as a negative control. Intensity of staining for ras p21 (Ip21) was expressed as the ratio of the peak channel numbers of the peak generated by staining with anti-ras p21 to the peak obtained by staining with an isotype control. Using this method, 21 out of 32 clinical specimens of acute leukemia were found to express ras p21 in elevated amounts compared to normal peripheral blood. Flow cytometry appears to be a practical method for routine screening of clinical specimens for the expression of oncogene products on individual cells rather than cell homogenates.

Combined parenteral and oral immunization results in an enhanced mucosal immunoglobulin A response to Shigella flexneri.

Achieving a vigorous secretory immunoglobulin A (IgA) response in intestinal secretions usually requires multiple doses of antigen given orally, while systemic immunity is more easily attained by parenteral immunization. This study examines the role of combined parenteral and oral immunizations to enhance the early mucosal immune response to an enteropathogen. We have used a chronically isolated intestinal-loop model in rabbits as a probe to monitor kinetically the initial (primary) local immune response to shigella lipopolysaccharide (LPS) following combinations of parenteral immunization intramuscularly (i.m.) and oral stimulation with shigellae. Predictably, effective stimulation of systemic immunity was elicited when heat-killed preparations of Shigella sp. strain X16 were given i.m., as shown by strong serum IgG and weak intestinal IgA activity to shigella LPS. A single oral dose of live Shigella sp. strain X16 given to unprimed rabbits elicited only a typical weak IgA response in intestinal secretions. However, when an i.m. dose of heat-killed shigellae was followed 1 day later by an oral dose of live Shigella sp. strain X16, a hyperstimulation of the early secretory IgA response was elicited, and the response reached levels found previously only after multiple oral administrations of live shigellae. This stimulation did not require the use of an adjuvant. At the same time, the animals receiving this combined oral and i.m. regimen had a lower IgG antishigella LPS activity in serum compared with their response after receiving parenteral antigen in adjuvant alone. These findings indicate that while a dichotomy exists between the systemic and mucosal immune responses, careful orchestration of the stimulatory events can promote a vigorous early local IgA response.