Treatment of ischemic wounds using cultured dermal fibroblasts transduced retrovirally with PDGF-B and VEGF121 genes.

The healing of ischemic wounds is a particularly difficult clinical challenge. In this study, rabbit dermal fibroblasts transduced retrovirally with human platelet-derived growth factor B (PDGF-B) and human vascular endothelial growth factor 121 (VEGF121) genes were used to treat wounds in a rabbit ischemic ear model. The PDGF-B and VEGF121 genes were obtained from human umbilical vein endothelial cells (HUVECs) by reverse transcription-polymerase chain reaction, cloned into retroviral vectors under control of the beta-actin promoter, and introduced into primary rabbit dermal fibroblast cells. In vitro results demonstrated that rabbit dermal fibroblasts are transduced and selected readily using retroviral vectors, and are engineered to secrete PDGF-B and VEGF121 at steady-state levels of 150 ng per 10(6) cells per 24 hours and 230 ng per 10(6) cells per 24 hours respectively. These cells were then seeded onto polyglycolic acid (PGA) scaffold matrices and used to treat ischemic rabbit ear wounds. Immunohistochemistry showed intense staining for PDGF-B and VEGF121 in the wounds treated with these transduced cells compared with the control treatment groups. For the relatively more ischemic distal ear wounds, granulation tissue deposition was increased significantly in the wounds treated with PDGF-B- and VEGF121-transduced cells compared with wounds treated with PGA alone. These results demonstrate that gene augmentation of rabbit dermal fibroblasts with the PDGF-B and VEGF121 genes introduced into this ischemic wound model via PGA matrices modulates wound healing, and may have clinical potential in the treatment of ischemic wounds.

Cartilage tissue engineering: current limitations and solutions.

Articular cartilage repair remains one of the most intensely studied orthopaedic topics. To date the field of tissue engineering has ushered in new methodologies for the treatment of cartilage defects. The authors' 10-year experience using principles of tissue engineering applied to resurfacing of cartilage defects is reported. Which cell type to use, chondrocytes versus chondroprogenitor cells, and their inherent advantages and disadvantages are discussed. Chondrocytes initially were used as the preferred cell type but were shown to have long term disadvantages in models used by the authors. Mesenchymal stem cells can be used effectively to overcome the limitations experienced with the use of differentiated chondrocytes. The use of mesenchymal stem cells as platforms for retroviral transduction of genes useful in cartilage repair introduces the concept of gene modified tissue engineering. The fundamental conditions for promoting and conducting a viable cartilage repair tissue, regardless of which cell type is used, also were studied. Placement of a synthetic porous biodegradable polymer scaffold was found to be a requirement for achieving an organized repair capable of functionally resurfacing a cartilage defect. A new modular device for intraarticular fixation of various graft composites has been developed. This new cartilage repair device is composed of bioabsorbable polymers and is capable of being delivered by the arthroscope.

Altered expression of the retinoblastoma susceptibility gene in chronic lymphocytic leukaemia.

The pathogenesis of chronic lymphocytic leukaemia (CLL) is unknown. One of the most frequent cytogenetic abnormalities in CLL is a deletion within the long arm of chromosome 13, the region to which the retinoblastoma (Rb) gene has been mapped. Lack of Rb expression has been linked to the carcinogenic process in many human tumours. We therefore sought to investigate the role of Rb gene inactivation in CLL using differential polymerase chain reaction on reverse transcribed RNA. The result of the PCR was quantitated using HPLC. 5/39 patients revealed a lack or significantly impaired expression of the Rb gene upon differential PCR analysis. In addition, immunocytochemical studies were performed using the Rb-specific monoclonal antibody PMG245. 10/56 patients showed a weak or absent expression upon immunocytochemical analysis compared to monocytes or granulocytes. The samples lacking Rb were from both early and late stage CLL. Our results indicate that inactivation of the Rb protein occurs in a fraction of CLL cases and can be found in early and late stages of the disease.

Evidence of cross-reactivity of 'carcinoma-specific' KC4 monoclonal antibody with activated mesothelial cells and phytohemagglutinin-stimulated lymphocytes.

Carcinoma-specific antibodies would be a useful tool in immunocytology of serous effusions. We tested the carcinoma-'specific' monoclonal antibody KC4 with cells obtained from pleural effusions evaluated by thoracoscopy and pleural biopsies. KC4 reacted most strongly with carcinoma cells. However, activated mesothelial cells also expressed this marker strongly. Lymphocytes stimulated with phytohemagglutinin were also stained by KC4. Thus KC4 appears to detect a proliferation antigen rather than a carcinoma-specific antigen.

[Effect of repeated leukaphereses on the lymphocytogram of blood donors]. / Einfluss von wiederholten Leukozytapheresen auf das Lymphozytogramm von Blutspendern.

Donors risk lymphopenia as a result of thrombocytapheresis as well as leukapheresis. The importance of the removal of lymphocytes is speculative at present. In the study presented, 10 healthy blood donors underwent 5 consecutive, discontinuous flow-leukapheresis procedures in weekly intervals with the effect of a total loss of 2.6 X 10(10) lymphocytes. The number of peripheral lymphocytes decreased steadily from 1,850 +/- 417/microliter to 1,451 +/- 411/microliter over the 5-week period. A significant decrease comprised also OKT3+-, OKT4+- and B-lymphocytes but not OKT8+-cells. Accordingly the OKT4/OKT8 ratio was reduced though not significantly from 1.41 +/- 0.25 to 1.32 +/- 0.27. 6 months after the last apheresis, the total lymphocyte count, OKT3+- and B-lymphocytes were still significantly low. The long-lasting peripheral lymphopenia in cytapheresis donors has to be taken into account as one of the criteria for donor selection. Donors with less than 1,000 lymphocytes/microliter of peripheral blood should be excluded from cytapheresis procedures.

Assessment of the beta-adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response.

We developed methods for identifying beta-adrenergic receptors in human right ventricular endomyocardial biopsy tissue with the radioligand (-)[125I]iodocyanopindolol (ICYP). Specific ICYP binding in a crude, high-yield membrane preparation derived from endomyocardial biopsy tissue was high (specificity greater than 90%), of high affinity (KD around 20 pM), saturable and stereospecific for the (-) vs the (+) isomer of isoproterenol. Subjects with mild-moderate and severe biventricular dysfunction had respective decreases in beta-adrenergic receptor density of 38.2% and 57.7% when normalization methods were averaged, with no significant differences in ICYP dissociation constant. A subgroup of subjects was subdivided by left ventricular ejection fraction (LVEF) into those with mild cardiac dysfunction (LVEF less than 0.50 greater than 0.40) and severe heart failure (LVEF less than 0.20) and given graded sequential infusions of dobutamine and calcium gluconate. Those with severe cardiac dysfunction had marked impairment of the dobutamine dP/dt and stroke work index response, whereas these responses to calcium did not differ in the two groups. These data indicate that in the intact human heart endomyocardial biopsy may be used for direct analysis of beta-adrenergic receptors, heart failure-associated myocardial beta-adrenergic down-regulation begins with mild-moderate ventricular dysfunction, reduction in myocardial beta-receptor density is related to degree of heart failure, and beta-receptor down-regulation is associated with pharmacologically specific impairment of the beta-agonist-mediated contractile response.

Endomyocardial biopsy.

Endomyocardial biopsy is an accepted, useful invasive tool for the analysis of human endomyocardium at the cellular and subcellular levels. It is applicable in the evaluation of specific diseases including cardiac allograft rejection, myocarditis, anthracycline cardiotoxicity, and infiltrative cardiomyopathies. The procedure can be performed in a cardiac catheterization room on an outpatient basis. The technique is quite safe when performed by trained cardiologists. Left ventricular biopsies are also safe but require systemic heparinization to prevent thromboembolization. The clinical indications for performing an endomyocardial biopsy include routine followup and suspected rejection of cardiac allograft, suspected myocarditis, monitoring or diagnosis of suspected anthracycline cardiotoxicity, and suspected secondary cardiomyopathies. Left ventricular endomyocardial biopsy is indicated for diseases that predominantly involve the left side of the heart, including left heart irradiation, cardiac fibroelastosis in infants, endomyocardial fibrosis, and scleroderma heart disease, and when right ventricular biopsy is unsuccessful. Endomyocardial biopsy is increasingly being used for research in the areas of tissue biochemistry, primary and valvular cardiomyopathies, immunology, beta receptor enzymology, drug interactions, and myocardial fibrosis. Endomyocardial biopsy has not been shown to be clinically useful in the evaluation of primary, dilated, hypertrophic, or alcoholic cardiomyopathies. These disease processes all lack pathognomomic microscopic abnormalities, and subclassification has neither been successful nor therapeutically useful. In addition, this technique is limited in diagnosing any cardiac abnormality that is not diffuse, inasmuch as only a few samples of the endomyocardial layer are obtained for evaluation. Therefore, a negative biopsy result is not 100 percent specific in excluding certain diseases. A further limitation of this technique is the need for an experienced cardiac pathologist who is well versed in interpretation of biopsy specimens. Finally, there should be a sufficiently large case load to train and to maintain skilled practitioners so that the procedure can be performed with little risk. The role of endomyocardial biopsy will continue to expand as research continues to find more uses for the technique and as more clinicians become skilled in its use.

Tissue response selectivity of calcium antagonists is not due to heterogeneity of [3H]-nitrendipine binding sites.

[3H]-nitrendipine binding data and isolated tissue response for five calcium antagonists were evaluated in rabbit myocardium and aorta. The [3H]-nitrendipine binding site was qualitatively identical in myocardium and aorta, as the [3H]-nitrendipine KD, KIS for nicardipine and nifedipine and interactions with verapamil, D600 and diltiazem were not different in aortic and cardiac membranes prepared by similar means. In contrast, the inhibition of the Ca2+-induced contractile response in right ventricular myocardium and aortic ring segments indicated a greater than 10,000 fold selectivity of nicardipine for antagonism of vascular responses. This resulted in a different order of potency for calcium antagonist interaction with the [3H]-nitrendipine binding site in cardiac membranes (nicardipine greater than nifedipine greater than D600 greater than verapamil greater than diltiazem) as compared to antagonism of myocardial tissue response (D600 greater than verapamil greater than or equal to nifedipine greater than nicardipine greater than or equal to diltiazem). In heart the difference between the potency of nicardipine in binding experiments and tissue response approached 4 orders of magnitude. We conclude that tissue response selectivity of calcium antagonists is not explained by heterogeneity of [3H]-nitrendipine binding sites.

Meningitis caused by maltose-negative variant of Neisseria meningitidis.

A maltose-negative variant of Neisseria meningitidis, Slaterus Y, was recovered from a patient with meningitis. A report of the case is presented and the medical-legal significance of such an isolate is briefly discussed.