The Current Role of Stem Cells in Orthopaedic Surgery.

Basic science and experimental research on stem cells has increased exponentially in the last decade. Our present knowledge about stem cell biology is better than ever before. This new paradigm shift in research has been reflected in the field of orthopaedic surgery. Various experimental models have suggested a potential application of stem cells for different orthopaedic conditions, and early clinical results of stem cell use have been encouraging. These cells can be easily isolated, processed and made available for clinical use. From healing of bone defects caused by trauma, tumor or infection to cartilage defects, nerve, tendon and ligament healing, stem cell use has the potential to revolutionize orthopaedic practice. The purpose of this article is to orient a general orthopaedic surgeon towards the current use and clinical applications of stem cell based therapy in orthopaedics and to provide a complete overview of the clinical advances in this field.

Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions.

The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically inhibited in their cytotoxicity by HLA-Cw*0304 and polyclonal human NK cells were used for conjugate formation with target cells that are either protected or are susceptible to NK cell-mediated cytotoxicity. Polarization of talin, microtubule-organizing center, and lysosomes occurred only during cytolytic interactions. The NK immune synapses were analyzed by three-dimensional immunofluorescence microscopy, which showed two distinctly different synaptic organizations in NK cells during cytolytic and noncytolytic interactions. The center of a cytolytic synapse with MHC class I-deficient target is comprised of a complex of signaling molecules including Src homology (SH)2-containing protein tyrosine phosphatase-1 (SHP-1). Closely related molecules with overlapping functions, such as the Syk kinases, SYK, and ZAP-70, and adaptor molecules, SH2 domain-containing leukocyte protein of 76 kDa and B cell linker protein, are expressed in activated NK cells and are all recruited to the center of the cytolytic synapse. In contrast, the noncytolytic synapse contains SHP-1, but is lacking other components of the central supramolecular activation cluster. These findings indicate a functional role for SHP-1 in both the cytolytic and noncytolytic interactions. We also demonstrate, in three-cell conjugates, that a single NK cell forms a cytolytic synapse with a susceptible target cell in the presence of both susceptible and nonsusceptible target cells.

Surgical strategy of complex empyema thoracis.

BACKGROUND: The optimal treatment of empyema thoracis has been widely debated. Proponents of pleural drainage alone, drainage plus fibrinolytic therapy, video-assisted thoracoscopic surgical (VATS) debridement, and open thoracotomy each champion the efficacy of their approach. METHODS: This study examines treatment of complex empyema thoracis between June 1, 1994, and April 30, 1997. Twenty-one men and 9 women underwent 30 drainage/decortication procedures (14 open thoracotomies and 16 VATS) in treatment of their disease. Effusion etiology was distributed as follows: infectious-14; neoplastic-associated-7; traumatic-3; other-6. RESULTS: The mean preoperative hospital stay was 14 +/- 8.8 days, (11.4 +/- 6.5 days for VATS vs 16.8 +/- 10.2 days for thoracotomy). Hospital stay from operation to discharge for thoracotomy patients was 10.0 -/+ 7.2 days (median 8.5 days) and for VATS patients 17.6 -/+ 16.8 days (median 11 days). These differences were not statistically significant. Duration of postoperative thoracostomy tube drainage was 8.3 -/+ 4.6 days for thoracotomy patients and 4.7 -/+ 2.8 days in the VATS group (p = 0.01). Operative time for the thoracotomy group was 125.0 -/+ 71.7 minutes, while the VATS group time was only 76.2 -/+ 30.7 minutes. Estimated blood loss for the thoracotomy group was 313.9 -/+ 254.0 milliliters and for the VATS group 131.6 -/+ 77.3 milliliters. Three of the 30 patients (10.0%) required prolonged ventilator support (>24 hours). Morbidity included one diaphragmatic laceration (VATS group) and one thoracic duct laceration (thoracotomy). Two VATS procedures (6.7%) required conversion to open thoracotomy for thorough decortication. CONCLUSIONS: The surgical approach to empyema thoracis is evolving. In the absence of comorbid factors, the significantly lower requirement for chest tube drainage time in the VATS patients suggests that this modality is an attractive alternative to thoracotomy in the treatment of complex empyema thoracis.

The role of cardioplegia induction temperature and amino acid enrichment in neonatal myocardial protection.

BACKGROUND: Warm cardioplegic induction improves the ischemically "stressed" adult heart. However, it is rarely used in infants, despite the fact that many newborn hearts are stressed by other factors such as hypoxia. The need for amino acids as well as their mechanism of action has also not been studied. METHODS: We first assessed the role of cardioplegic induction temperature in 10 nonhypoxic neonatal piglets undergoing 70 minutes of multidose blood cardioplegic arrest. Five piglets (group 1) received a cold (4 degrees C) induction, and 5 (group 2) a warm (37 degrees C) induction. Twenty-six other piglets underwent ventilator hypoxia (fraction of inspired oxygen, 8% to 10%) for 60 minutes before cardiopulmonary bypass (stress). Six piglets (group 3) then underwent 70 minutes of cardiopulmonary bypass without ischemia (hypoxia controls), and 20 underwent 70 minutes of cardioplegic arrest. Five of these (group 4) received cold cardioplegic induction, and 15 received warm induction; in 5 of these (group 5), the warm cardioplegic solution contained amino acids, in 5 others (group 6), it was unsupplemented, and in the remaining 5 (group 7), nitroglycerin was added to determine the role of vasodilation. Myocardial function was assessed by pressure-volume loops (expressed as a percent of control), and coronary vascular resistance was measured with cardioplegic infusions. RESULTS: In nonhypoxic (normal) piglets, cold (group 1) and warm (group 2) induction completely preserved systolic function (end-systolic elastance, 100% versus 104%) and preload recruitable stroke work (100% versus 102%), with minimal increase in diastolic compliance (162% versus 156%). Hypoxia-reoxygenation alone (group 3) depressed systolic function (end-systolic elastance, 51%+/-2%) and preload recruitable stroke work (54%+/-3%), and raised diastolic stiffness (260%+/-15%). The detrimental effects of reoxygenation persisted (unchanged from reoxygenation alone) with cold induction (group 4) or warm induction without amino acids (groups 6 and 7). In contrast, warm induction with amino acids (group 5) restored systolic function (end-systolic elastance, 105%+/-3%; p < 0.001 versus groups 3, 4, 6, and 7) and preload recruitable stroke work (103%+/-2%; p < 0.001 versus groups 3, 4, 6, and 7), and decreased diastolic stiffness (154%+/-7%; p < 0.001 versus groups 3, 4, 6, and 7). However, there was no difference in myocardial oxygen consumption in hypoxic hearts receiving a warm induction (6.9 versus 6.5 versus 7.3 mL/g per 5 minutes) (groups 5, 6, 7), and coronary vascular resistance was lowest with nitroglycerin (group 7). CONCLUSIONS: Cardioplegic induction can be given either warm or cold in nonhypoxic neonatal hearts. In contrast, only warm induction with amino acids repairs the hypoxic injury, but the primary mechanism of action is not related to increased metabolic activity or vasodilation.

Roles of replication protein-A subunits 2 and 3 in DNA replication fork movement in Saccharomyces cerevisiae.

Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants. Thus, replication fork movement in vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode.

Assessing the requirements for nucleotide excision repair proteins of Saccharomyces cerevisiae in an in vitro system.

Nucleotide excision repair (NER) is the primary mechanism by which both Saccharomyces cerevisiae and human cells remove the DNA lesions caused by ultraviolet light and other mutagens. This complex process involves the coordinated actions of more than 20 polypeptides. To facilitate biochemical studies of NER in yeast, we have established a simple protocol for preparing whole cell extracts which perform NER in vitro. As expected, this assay of in vitro repair was dependent on the products of RAD genes such as RAD14, RAD4, and RAD2. Interestingly, it was also dependent upon proteins encoded by the RAD7, RAD16, and RAD23 genes whose precise roles in NER are uncertain, but not the RAD26 gene whose product is believed to participate in coupling NER to transcription. Replication protein A (RPA/Rpa), known to be required for NER in human cell extracts, was also shown by antibody inhibition and immunodepletion experiments to be required for NER in our yeast cell extracts. Moreover, yeast cells with temperature-sensitive mutations in the RFA2 gene, which encodes the 34-kDa subunit of Rpa, had increased sensitivity to UV and yielded extracts defective in NER in vitro. These data indicate that Rpa is an essential component of the NER machinery in S. cerevisiae as it is in mammalian cells.

A hierarchy of SSB protomers in replication protein A.

Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.

Structural basis for X-linked agammaglobulinemia (XLA): mutations at interacting Btk residues R562, W563, and A582.

It has been suggested that tryptophan 563 is sandwiched between residues R562 and A582 in Bruton's agammaglobulinemia tyrosine kinase (Btk). Mutations of the surrounding residues have been shown to cause X-linked agammaglobulinemia. Substitutions R562P and A582V were noticed to have impaired kinase activity. However, based on Western blot analysis, the mutant proteins were expressed at normal levels. Molecular modeling of the kinase domain has previously indicated that these residues presumably govern the position of the W563 side chain, which is thought to interact with the catalytic loop. W563 is inside the molecule and too far away from the catalytic center to interact directly with the substrate or cofactors. To prove these model-based conclusions, a conservative substitution with phenylalanine for W563 was made, and the resultant mutant lacked kinase activity. These results confirm our previous assumption that the side chain of W563, invariant in protein tyrosine kinases, is crucial for Btk kinase activity. Mutations in the surrounding residues seem to inactivate Btk by affecting the location of W563.

Structural basis for chromosome X-linked agammaglobulinemia: a tyrosine kinase disease.

X-linked agammaglobulinemia (XLA) is a hereditary defect of B-cell differentiation in man caused by deficiency of Bruton tyrosine kinase (BTK). A three-dimensional model for the BTK kinase domain, based on the core structure of cAMP-dependent protein kinase, was used to interpret the structural basis for disease in eight independent point mutations in patients with XLA. As Arg-525 of BTK has been thought to functionally substitute for a critical lysine residue in protein-serine kinases, the mutation Arg-525-->Gln was studied and found to abrogate the tyrosine kinase activity of BTK. All of the eight mutations (Lys-430-->Glu, Arg-520-->Glu, Arg-525-->Gln, Arg-562-->Pro, Ala-582-->Val, Glu-589-->Gly, Gly-594-->Glu, and Gly-613-->Asp) were located on one face of the BTK kinase domain, indicating structural clustering of functionally important residues.

Expression of Bruton's agammaglobulinemia tyrosine kinase gene, BTK, is selectively down-regulated in T lymphocytes and plasma cells.

The gene mutated in the human disease, X-linked agammaglobulinemia (XLA), is related to the Src gene family of cytoplasmic protein-tyrosine kinases and is designated Btk (Bruton's agammaglobulinemia tyrosine kinase; formerly Atk/Bpk; the human gene is denoted BTK, using capital letters according to the kinase nomenclature). We have recently reported that this gene is expressed in B lymphocytes and that the specific mRNA was undetectable in T cells using Northern blotting. Further analyses of different sources of B and T lymphocytes confirmed this pattern. However, BTK transcripts were undetectable in four plasmacytoma lines. Moreover, as virtually normal amounts of BTK transcripts were found in PBMC from two patients carrying a point mutation in BTK, despite low B cell numbers, we anticipated that the gene would also be expressed in cells of other lineages. The erythroleukemia cell line K-562, the promyelocytic line HL-60 and the histiocytic lymphoma line U-937 were found to have BTK mRNA levels comparable to B cells. BTK mRNA was also detected in monocytes from healthy donors as well as in the human immature basophilic cell line KU812, in the human mast cell leukemia cell line HMC-1 and in the CD34 expressing myeloblast KG-1. A similar expression pattern was obtained when BTK protein was analyzed by immunoprecipitation and Western blotting. Using a polymerase chain reaction-based analysis, a small amount (less than 1% of the level in B cells) of BTK mRNA was identified in T lymphocytes. Our findings are compatible with a general expression of the BTK gene in hematopoietic cells, except in T lymphocytes and plasma cells, in which the transcript level is selectively down-regulated.

Modulation of tumour associated antigen expressed on human squamous cell carcinoma cell lines by recombinant interferon-alpha.

Our earlier studies have shown that monoclonal antibody (Mab) 3F8E3 generated against a head and neck cancer cell line LICR-LON-HN2 showed reactivity with squamous cell carcinoma (SCC) irrespective of the tissue of origin and identified antigens on SCC cell lines AW 13516 and AW 8507. The affinity constants (Ka) for binding of Mab 3F8E3 to AW 13516 and AW 8507 cell lines were 6.2 x 10(8) and 4.3 x 10(8) mol/l and it identified 6.8 x 10(4) and 3.77 x 10(4) sites/cell, respectively, as determined by Scatchard analysis. Treatment of both the cell lines with recombinant human interferon-alpha (rHu-IFN alpha) increased the binding affinity of the Mab but did not increase the number of binding sites on the SCC cell lines. Shedding of antigen recognised by the Mab in the culture supernatant of the cell lines was reduced after rHu-IFN alpha treatment. The results suggest that rHu-IFN alpha may bring about a firm anchorage of the tumour associated antigen on the SCC cells. Cells modulated with rHu-IFN alpha may serve as better targets for assessing cell mediated as well as Mab mediated cytotoxicity in oral cancer patients.

Modulatory effect of cytokines on natural killer and antibody dependent cellular cytotoxicity directed to squamous cell carcinoma targets by lymphocytes from oral cancer patients.

Cells from solid tumours are generally poor targets for natural killer (NK) cytotoxicity and antibody dependent cellular cytotoxicity (ADCC). In this paper, we have analysed NK cytotoxicity and ADCC mediated by peripheral blood mononuclear cells from healthy individuals and oral cancer patients before and after modulation with recombinant interleukin-2 (rIL-2), on target cells derived from two squamous cell carcinoma (SCC) cell lines prior to and after treatment with recombinant interferon-alpha (rIFN alpha). Target SCC cell directed monoclonal antibody 3F8E3 was used in ADCC. The results showed that the unmodulated SCC cells were poor targets for NK and ADCC compared to standard targets (K562 cells and chicken red blood cells, respectively). Modulation of targets alone with rIFN alpha showed moderate increase in their susceptibility while rIL-2 treated effectors could significantly lyse even unmodulated targets. Combined treatment of targets with rIFN alpha and effectors with rIL-2 showed additive enhancement in NK and ADCC activity against SCC cells. Lymphocytes from treated patients with recurrent disease could not efficiently lyse SCC targets even after combined modulation.

Antibody dependent cellular cytotoxicity and complement mediated cytotoxicity on leukemic cells mediated by anti K562 monoclonal antibodies.

Three anti K562 monoclonal antibodies (MAb) 4.3, 4.6 and 4.8 reacting predominantly with cells of myeloid lineage, were tested for antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). MAb 4.6 (IgG3k) effectively mediated ADCC against K562 cells and fresh leukemic targets with effectors from healthy donors. However, for ADCC on chronic myeloid leukemia (CML) targets, effectors from CML patients in remission needed modulation with IL-2. All MAb showed significant CDC against peripheral blood (PB) and bone marrow (BM) cells obtained from CML patients in chronic phase, and untreated acute myeloid leukemia (AML) patients. MAb displayed no CDC against PB and BM cells from CML patients in remission and BM cells of Hodgkin's Disease (HD) patients with normal BM cellularity. In clonogenic assay, colony forming units (CFU) in the BM aspirate obtained from CML patients in chronic phase were significantly reduced by treatment with MAb and complement.

Immunoreactivity of anti K562 monoclonal antibodies with leukemic cells of myeloid lineage.

Three monoclonal antibodies (MAb) raised against K562 cells (erythroleukemic cell line) reacting specifically with myeloid maturation related antigens, were characterized. MAb 4.3 (IgG2bk) reacted with promyelocytes, metamyelocytes and myelocytes, MAb 4.6 (IgG3k) also identified the myeloid blasts, while MAb 4.8 (IgG1k) reacted with metamyelocytes. The affinity constant of the MAbs ranged from 0.8 X 10(7) -3.9 X 10(8) M-1 and the binding sites on K562 cells varied from 8 X 10(4)-4 X 10(5). In competition RIA MAbs 4.6 and 4.8 competed with each other for binding sites on K562 cells. In Western blot analysis the MAbs reacted with 66,000 mol.wt and 84,000 mol.wt peptides on K562 cells and 97,000 mol.wt peptide on chronic myeloid leukemia (CML) cells. These MAbs may help in immunophenotyping of myeloid leukemias.

Monoclonal antibody against human squamous-cell-carcinoma-associated antigen.

Mouse monoclonal antibody (MAb) 3F8E3 (IgG3k) was developed against the head and neck cancer cell line LICR-LON-HN2. Subjected to indirect immunofluorescence, the MAb reacted exclusively with SCC cell lines and showed no reactivity with normal or transformed mouse and human non-SCC cell lines and hematopoietic cell lines. The radiolabelled MAb showed an affinity constant of 1.8 x 10(8) M-1 with HN2 cells and identified 2.07 x 10(4) sites/cell by Scatchard analysis. It identified 2 peptides from membrane extracts of HN2 cells by Western blotting. Avidin-biotin-complexed immunoperoxidase staining on cryostat sections of tumors from various tissues revealed that 3F8E3 reacted mainly with the membrane antigens of well differentiated SCC cells of oral cavity, larynx, esophagus, lung, uterine cervix, metastatic nodes of patients with oral cancer, and dysplastic cells in oral leukoplakia. The MAb did not react with poorly differentiated cells of Ca esophagus, adenocarcinoma of breast, stomach and colon, renal-cell carcinoma and soft-tissue sarcoma.

Radioprotection of euoxic bacteria by phenothiazine drugs.

Survival studies on irradiated euoxic E. coli B/r cells in presence of various concentrations of four radioprotecting phenothiazine drugs have been carried out. Maximum radioprotection was obtained at a optimal concentration for each drug and it decreased on either side of it. The DNA strand break studies at the maximum protective and non-protective concentrations of chlorpromazine and promethazine revealed that the number of ssbs in DNA were less at the protective concentration which were efficiently repaired by the type-III repair process. On the other hand, at the non-protective concentrations, inhibition of DNA repair was noticed and a higher number of DNA ssbs were detected. We suggest that the membrane is fluidized to a greater extent at the protective concentrations allowing the chemical restitution of damaged sites by NPSH compounds. At the non-protective high concentrations of the drugs, the membrane may be too grossly disorganised to allow any repair and at the same time high concentrations of the drugs or their radicals may also react with radioprotective intracellular sulphhydryls.

Modification of radiation response of E. coli B/r cells by phenothiazines.

Promethazine and trimeprazine sensitized anoxic E. coli B/r cells to 60Co gamma-rays, but both drugs showed a radioprotective effect under euoxic conditions. Their radiosensitizing effect was found to be due to the reaction of radiolytically induced hydroxyl radicals with the sensitizers. The radioprotective effect of these drugs is attributed to changes in the membrane structure conducive with chemical repair of the damaged sites in the gel region of the cellular membrane by intracellular sulphydryl compounds. Pre-irradiation depletion of sulphydryls from E. coli B/r by treatment with N-ethyl maleimide abolished the radio-protective effect of these drugs under euoxic conditions.