text:
CDR-grafted antibody heavy and light chains comprise acceptor framework and donor antigen binding regions, the heavy chains comprising donor residues at at least one of positions (6, 23) and/or (24, 48) and/or (49, 71) and/or (73, 75) and/or (76) and/or (78) and (88) and/or (91). The CDR-grafted light chains comprise donor residues at at least one of positions (1) and/or (3) and (46) and/or (47) or at at least one of positons (46, 48, 58) and (71). The CDR-grafted antibodies are preferably humanized antibodies, having non human e.g. rodent, donor and human acceptor frameworks, and may be used for in vivo therapy and diagnosis. A generally applicable protocol is disclosed for obtaining CDR-grafted antibodies.
1. A host cell tranformed with a nucleic acid encoding an antibody molecule having affinity for a predetermined antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs) and framework regions, wherein said framework regions of said variable domain comprise predominantly human acceptor antibody heavy chain framework region residues, the remaining heavy chain framework region residues corresponding to the equivalent residues in a donor antibody having affinity for said predetermined antigen, wherein, according to the Kabat numbering system, in said composite heavy chain, said CDRs comprise donor residues at residues 31 to 35, 50 to 58, and 95 to 102; and said framework regions comprise donor residues at amino acid residues 6, 24, 48, 49, 71, 73, and 78.
2. The host cell of
3. The host cell of
4. The host cell of
5. The host cell of
6. The host cell of
7. The host cell of
The present application is a Continuation of application Ser. No. 08/846,658, Filed on May 1, 1997, which is a Continuation of application Ser. No. 08/303,569, Filed on Sep. 7, 1994, now U.S. Pat. No. 5,859,205, Issued on Jan. 12, 1999, which is a File Wrapper Continuation of application Ser. No. 07/743,329, Filed on Sep. 17, 1991, Abandoned, which is a 371 National Phase Filing of PCT/GB90/02017, International Filing Date of Dec. 21, 1990, which claims priority to GB 8928874.0, Filed on Dec. 21, 1989, all applications incorporated by reference herein in their entireties.
The present invention relates to humanised antibody molecules, to processes for their production using recombinant DNA technology, and to their therapeutic uses.
The term �humanised antibody molecule� is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, and remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The antigen binding site typically comprises complementarity determining regions (CDRs) which determine the binding specificity of the antibody molecule and which are carried on appropriate framework regions in the variable domains. There are 3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light chain variable domains.
In the description, reference is made to a number of publications by number. The publications are listed in numerical order at the end of the description.
Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, (Fab′)2 and Fc fragments, which can be derived by enzymatic cleavage. Natural immunoglobulins comprise a generally Y-shaped molecule having an antigen-binding site towards the end of each upper arm. The remainder of the structure, and particularly the stem of the Y, mediates the effector functions associated with immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to a more limited extent, therapy. However, such uses, especially in therapy, were hindered until recently by the polyclonal nature of natural immunoglobulins. A significant step towards the realisation of the potential of immunoglobulins as therapeutic agents was the discovery of procedures for the production of monoclonal antibodies (MAbs) of defined specificity (1).
However, most MAbs are produced by hybridomas which are fusions of rodent spleen cells with rodent myeloma cells. They are therefore essentially rodent proteins. There are very few reports of the production of human MAbs.
Since most available MAbs are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the MAb and will either remove it entirely or at least reduce its effectiveness. In practice, MAbs of rodent origin may not be used in patients for more than one or a few treatments as a HAMA response soon develops rendering the MAb ineffective as well as giving rise to undesirable reactions. For instance, OKT3 a mouse IgG2a/k MAb which recognises an antigen in the T-cell receptor-CD3 complex has been approved for use in many countries throughout the world as an immunosuppressant in the treatment of acute allograft rejection [Chatenoud et al (2) and Jeffers et al (3)]. However, in view of the rodent nature of this and other such MAbs, a significant HAMA response which may include a major anti-idiotype component, may build up on use. Clearly, it would be highly desirable to diminish or abolish this undesirable HAMA response and thus enlarge the areas of use of these very useful antibodies.
Proposals have therefore been made to render non-human MAbs less antigenic in humans. Such techniques can be generically termed �humanisation� techniques. These techniques typically involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
Early methods for humanizing MAbs involved production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody is linked to constant domains derived from another antibody. Methods for carrying out such chimerisation procedures are described in EP0120694 (Celltech Limited), EP0125023 (Genentech Inc. and City of Hope), EP-A-0 171496 (Res. Dev. Corp. Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (Celltech Limited). This latter Celltech application (WO 86/01533) discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin. Such humanised chimeric antibodies, however, still contain a significant proportion of non-human amino acid sequence, i.e. the complete non-human variable domains, and thus may still elicit some HAMA response, particularly if administered over a prolonged period [Begent et al (ref. 4)].
In an alternative approach, described in EP-A-0239400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb have been grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides. The present invention relates to humanised antibody molecules prepared according to this alternative approach, i.e. CDR-grafted humanised antibody molecules. Such CDR-grafted humanised antibodies are much less likely to give rise to a HAMA response than humanised chimeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain.
The earliest work on humanising MAbs by CDR-grafting was carried out on MAbs recognizing synthetic antigens, such as the NP or NIP antigens. However, examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T-cells were humanised by CDR-grafting have been described by Verhoeyen et al (5) and Riechmann et al (6) respectively. The preparation of CDR-grafted antibody to the antigen on human T cells is also described in WO 89/07452 (Medical Research Council).
In Riechmann et al/Medical Research Council it was found that transfer of the CDR regions alone [as defined by Kabat refs. (7) and (8)] was not sufficient to provide satisfactory antigen binding activity in the CDR-grafted product. Riechmann et al found that it was necessary to convert a serine residue at position 27 of the human sequence to the corresponding rat phenylalanine residue to obtain a CDR-grafted product having improved antigen binding activity. This residue at position 27 of the heavy chain is within the structural loop adjacent to CDR1. A further construct which additionally contained a human serine to rat tyrosine change at position 30 of the heavy chain did not have a significantly altered binding activity over the humanised antibody with the serine to phenylalanine change at position 27 alone. These results indicate that changes to residues of the human sequence outside the CDR regions, in particular in the structural loop adjacent to CDR1, may be necessary to obtain effective antigen binding activity for CDR-grafted antibodies which recognise more complex antigens. Even so the binding affinity of the best CDR-grafted antibodies obtained was still significantly less than the original MAb.
Very recently Queen et al (9) have described the preparation of a humanised antibody that binds to the interleukin 2 receptor, by combining the CDRs of a murine MAb (anti-Tac) with human immunoglobulin framework and constant regions. The human framework regions were chosen to maximise homology with the anti-Tac MAb sequence. In addition computer modelling was used to identify framework amino acid residues which were likely to interact with the CDRs or antigen, and mouse amino acids were used at these positions in the humanized antibody.
In WO 90/07861 Queen et al propose four criteria for designing humanised immunoglobulins. The first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusually homologous to the non-human donor immunoglobulin to be humanised, or to use a consensus framework from many human antibodies. The second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework. The third criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs. The fourth criterion is to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a side chain atom within about 3 Å of the CDRs in a three-dimensional immunoglobulin model and to be capable of interacting with the antigen or with the CDRs of the humanised immunoglobulin. It is proposed that criteria two, three or four may be applied in addition or alternatively to criterion one, and may be applied singly or in any combination.
WO 90/07861 describes in detail the preparation of a single CDR-grafted humanised antibody, a humanised antibody having specificity for the p55 Tac protein of the IL-2 receptor. The combination of all four criteria, as above, were employed in designing this humanised antibody, the variable region frameworks of the human antibody Eu (7) being used as acceptor. In the resultant humanised antibody the donor CDRs were as defined by Kabat et al (7 and 8) and in addition the mouse donor residues were used in place of the human acceptor residues, at positions 27, 30, 48, 66, 67, 89, 91, 94, 103, 104, 105 and 107 in the heavy chain and at positions 48, 60 and 63 in the light chain, of the variable region frameworks. The humanized anti-Tac antibody obtained is reported to have an affinity for p55 of 3�109 M−1, about one-third of that of the murine MAb.
We have further investigated the preparation of CDR-grafted humanised antibody molecules and have identified a hierarchy of positions within the framework of the variable regions (i.e. outside both the Kabat CDRs and structural loops of the variable regions) at which the amino acid identities of the residues are important for obtaining CDR-grafted products with satisfactory binding affinity. This has enabled us to establish a protocol for obtaining satisfactory CDR-grafted products which may be applied very widely irrespective of the level of homology between the donor immunoglobulin and acceptor framework. The set of residues which we have identified as being of critical importance does not coincide with the residues identified by Queen et al (9).
Accordingly, in a first aspect the invention provides a CDR-grafted antibody heavy chain having a variable region domain comprising acceptor framework and donor antigen binding regions wherein the framework comprises donor residues at at least one of positions 6, 23 and/or 24, 48 and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/or 91.
In preferred embodiments, the heavy chain framework comprises donor residues at positions 23, 24, 49, 71, 73 and 78 or at positions 23, 24 and 49. The residues at positions 71, 73 and 78 of the heavy chain framework are preferably either all acceptor or all donor residues.
In particularly preferred embodiments the heavy chain framework additionally comprises donor residues at one, some or all of positions 6, 37, 48 and 94. Also it is particularly preferred that residues at positions of the heavy chain framework which are commonly conserved across species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106 and 107, if not conserved between donor and acceptor, additionally comprise donor residues. Most preferably the heavy chain framework additionally comprises donor residues at positions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
In addition the heavy chain framework optionally comprises donor residues at one, some or all of positions:
In the first and other aspects of the present invention reference is made to CDR-grafted antibody products comprising acceptor framework and donor antigen binding regions. It will be appreciated that the invention is widely applicable to the CDR-grafting of antibodies in general. Thus, the donor and acceptor antibodies may be derived from animals of the same species and even same antibody class or sub-class. More usually, however, the donor and acceptor antibodies are derived from animals of different species. Typically the donor antibody is a non-human antibody, such as a rodent MAb, and the acceptor antibody is a human antibody.
In the first and other aspects of the present invention, the donor antigen binding region typically comprises at least one CDR from the donor antibody. Usually the donor antigen binding region comprises at least two and preferably all three CDRs of each of the heavy chain and/or light chain variable regions. The CDRs may comprise the Kabat CDRs, the structural loop CDRs or a composite of the Kabat and structural loop CDRs and any combination of any of these. Preferably, the antigen binding regions of the CDR-grafted heavy chain variable domain comprise CDRs corresponding to the Kabat CDRs at CDR2 (residues 50�65) and CDR3 (residues 95�100) and a composite of the Kabat and structural loop CDRs at CDR1 (residues 26�35).
The residue designations given above and elsewhere in the present application are numbered according to the Kabat numbering [refs. (7) and (8)]. Thus the residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or CDR, of the basic variable domain structure. For example, the heavy chain variable region of the anti-Tac antibody described by Queen et al (9) contains a single amino acid insert (residue 52a) after residue 52 of CDR2 and a three amino acid insert (residues 82a, 82b and 82c) after framework residue 82, in the Kabat numbering. The correct Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a �standard� Kabat numbered sequenced.
The invention also provides in a second aspect a CDR-grafted antibody light chain having a variable region domain comprising acceptor framework and donor antigen binding regions wherein the framework comprises donor residues at at least one of positions 1 and/or 3 and 46 and/or 47. Preferably the CDR grafted light chain of the second aspect comprises donor residues at positions 46 and/or 47.
The invention also provides in a third aspect a CDR-grafted antibody light chain having a variable region domain comprising acceptor framework and donor antigen binding regions wherein the framework comprises donor residues at at least one of positions 46, 48, 58 and 71.
In a preferred embodiment of the third aspect, the framework comprises donor residues at all of positions 46, 48, 58 and 71.
In particularly preferred embodiments of the second and third aspects, the framework additionally comprises donor residues at positions 36, 44, 47, 85 and 87. Similarly positions of the light chain framework which are commonly conserved across species, i.e. positions 2, 4, 6, 35, 49, 62, 64�69, 98, 99, 101 and 102, if not conserved between donor and acceptor, additionally comprise donor residues. Most preferably the light chain framework additionally comprises donor residues at positions 2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64�69, 85, 87, 98, 99, 101 and 102.
In addition the framework of the second or third aspects optionally comprises donor residues at one, some or all of positions:
Preferably, the antigen binding regions of the CDR-grafted light chain variable domain comprise CDRs corresponding to the Kabat CDRs at CDR1 (residue 24�34), CDR2 (residues 50�56) and CDR3 (residues 89�97).
The invention further provides in a fourth aspect a CDR-grafted antibody molecule comprising at least one CDR-grafted heavy chain and at least one CDR-grafted light chain according to the first and second or first and third aspects of the invention.
The humanised antibody molecules and chains of the present invention may comprise: a complete antibody molecule, having full length heavy and light chains; a fragment thereof, such as a Fab, (Fab′)2 or FV fragment; a light chain or heavy chain monomer or diner; or a single chain antibody, e.g. a single chain FV in which heavy and light chain variable regions are joined by a peptide linker; or any other CDR-grafted molecule with the same specificity as the original donor antibody. Similarly the CDR-grafted heavy and light chain variable region may be combined with other antibody domains as appropriate.
Also the heavy or light chains or humanised antibody molecules of the present invention may have attached to them an effector or reporter molecule. For instance, it may have a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure. Alternatively, the procedures of recombinant DNA technology may be used to produce an immunoglobulin molecule in which the Fc fragment or CH3 domain of a complete immunoglobulin molecule has been replaced by, or has attached thereto by peptide linkage, a functional non-immunoglobulin protein, such as an enzyme or toxin molecule.
Any appropriate acceptor variable region framework sequences may be used having regard to class/type of the donor antibody from which the antigen binding regions are derived. Preferably, the type of acceptor framework used is of the same/similar class/type as the donor antibody. Conveniently, the framework may be chosen to maximise/optimise homology with the donor antibody sequence particularly at positions close or adjacent to the CDRs. However, a high level of homology between donor and acceptor sequences is not important for application of the present invention. The present invention identifies a hierarchy of framework residue positions at which donor residues may be important or desirable for obtaining a CDR-grafted antibody product having satisfactory binding properties. The CDR-grafted products usually have binding affinities of at least 105 M−1, preferably at least about 108 M−1, or especially in the range 108�1012 M−1. In principle, the present invention is applicable to any combination of donor and acceptor antibodies irrespective of the level of homology between their sequences. A protocol for applying the invention to any particular donor-acceptor antibody pair is given hereinafter. Examples of human frameworks which may be used are KOL, NEWM, REI, EU, LAY and POM (refs. 4 and 5) and the like; for instance KOL and NEWM for the heavy chain and REI for the light chain and EU, LAY and POM for both the heavy chain and the light chain.
Also the constant region domains of the products of the invention may be selected having regard to the proposed function of the antibody in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgE, IgG or IgM domains. In particular, IgG human constant region domains may be used, especially of the IgG1 and IgG3 isotypes, when the humanised antibody molecule is intended for therapeutic uses, and antibody effector functions are required. Alternatively, IgG2 and IgG4 isotypes may be used when the humanised antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, e.g. for simple blocking of lymphokine activity.
However, the remainder of the antibody molecules need not comprise only protein sequences from immunoglobulins. For instance, a gone may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding the amino acid sequence of a functional polypeptide such as an effector or reporter molecule.
Preferably the CDR-grafted antibody heavy and light chain and antibody molecule products are produced by recombinant DNA technology.
Thus in further aspects the invention also includes DNA sequences coding for the CDR-grafted heavy and light chains, cloning and expression vectors containing the DNA sequences, host cells transformed with the DNA sequences and processes for producing the CDR-grafted chains and antibody molecules comprising expressing the DNA sequences in the transformed host cells.
The general methods by which the vectors may be constructed, transfection methods and culture methods are well known per se and form no part of the invention. Such methods are shown, for instance, in references 10 and 11.
The DNA sequences which encode the donor amino acid sequence may be obtained by methods well known in the art. For example the donor coding sequences may be obtained by genomic cloning, or cDNA cloning from suitable hybridoma cell lines. Positive clones may be screened using appropriate probes for the heavy and light chain genes in question. Also PCR cloning may be used.
DNA coding for acceptor, e.g. human acceptor, sequences may be obtained in any appropriate way. For example DNA sequences coding for preferred human acceptor frameworks such as KOL, REI, EU and NEWM, are widely available to workers in the art.
The standard techniques of molecular biology may be used to prepare DNA sequences coding for the CDR-grafted products. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate. For example oligonucleotide directed synthesis as described by Jones et al (ref. 20) may be used. Also oligonucleotide directed mutagenesis of a pre-exising variable region as, for example, described by Verhoeyen et al (ref. 5) or Riechmann et al (ref. 6) may be used. Also enzymatic filling in of gapped oligonucleotides using T4 DNA polymerase as, for example, described by Queen et al (ref. 9) may be used.
Any suitable host cell/vector system may be used for expression of the DNA sequences coding for the CDR-grafted heavy and light chains. Bacterial e.g. E. coli, and other microbial systems may be used, in particular for expression of antibody fragments such as FAb and (Fab′)2 fragments, and especially FV fragments and single chain antibody fragments e.g. single chain FVs. Eucaryotic e.g. mammalian host cell expression system may be used for production of larger CDR-grafted antibody products, including complete antibody molecules. Suitable mammalian host calls include CEO cells and myeloma or hybridoma cell lines.
Thus, in a further aspect the present invention provides a process for producing a CDR-grafted antibody product comprising:
The CDR-grafted product may comprise only heavy or light chain derived polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence is used to transfect the host cells.
For production of products comprising both heavy and light chains, the cell line may be transfected with two vectors, the first vector may contain an operon encoding a light chain-derived polypeptide and the second vector containing an operon encoding a heavy chain-derived polypeptide. Preferably, the vectors are identical, except in so far as the coding sequences and selectable markers are concerned, so as to ensure as far as possible that each polypeptide chain is equally expressed. Alternatively, a single vector may be used, the vector including the sequences encoding both light chain- and heavy chain-derived polypeptides.
The DNA in the coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both. However, it is preferred that the DNA sequence encoding the heavy or light chain comprises at least partially, genomic DNA, preferably a fusion of cDNA and genomic DNA.
The present invention is applicable to antibodies of any appropriate specificity. Advantageously, however, the invention may be applied to the humanisation of non-human antibodies which are used for in vivo therapy or diagnosis. Thus the antibodies may be site-specific antibodies such as tumour-specific or cell surface-specific antibodies, suitable for use in in vivo therapy or diagnosis, e.g. tumour imaging. Examples of cell surface-specific antibodies are anti-T cell antibodies, such as anti-CD3, and CD4 and adhesion molecules, such as CR3, ICAM and ELAM. The antibodies may have specificity for interleukins (including lymphokines, growth factors and stimulating factors), hormones and other biologically active compounds, and receptors for any of these. For example, the antibodies may have specificity for any of the following: Interferons α, β, γ or δ, IL1, IL2, IL3, or IL4, etc., TNF, GCSF, GMCSF, EPO, hGH, or insulin, etc.
The the present invention also includes therapeutic and diagnostic compositions comprising the CDR-grafted products of the invention and uses of such compositions in therapy and diagnosis.
Accordingly in a further aspect the invention provides a therapeutic or diagnostic composition comprising a CDR-grafted antibody heavy or light chain or molecule according to previous aspects of the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.
Accordingly also the invention provides a method of therapy or diagnosis comprising administering an effective amount of a CDR-grafted antibody heavy or light chain or molecule according to previous aspects of the invention to a human or animal subject.
A preferred protocol for obtaining CDR-grafted antibody heavy and light chains in accordance with the present invention is set out below together with the rationale by which we have derived this protocol. This protocol and rationale are given without prejudice to the generality of the invention as hereinbefore described and defined.
It is first of all necessary to sequence the DNA coding for the heavy and light chain variable regions of the donor antibody, to determine their amino acid sequences. It is also necessary to choose appropriate acceptor heavy and light chain variable regions, of known amino acid sequence. The CDR-grafted chain is then designed starting from the basis of the acceptor sequence. It will be appreciated that in same cases the donor and acceptor amino acid residues may be identical at a particular position and thus no change of acceptor framework residue is required.
1. As a first step donor residues are substituted for acceptor residues in the CDRs. For this purpose the CDRs are preferably defined an follows:
In order to transfer the binding site of an antibody into a different acceptor framework, a number of factors need to be considered.
1. The Extent of the CDRs
The above analysis coupled with our considerable practical experimental experience in the CDR-grafting of a number of different antibodies have lead us to the protocol given above.
The present invention is now described, by way of example only, with reference to the accompanying
CDR-Grafting of OKT3
Material and Methods
1. Incoming Cells
The cDNA library was ligated to pSP65 vector DNA which had been EcoR1 cut and the 5′ phosphate groups removed by calf intestinal phosphatase (EcoR1/CIP). The ligation was used to transform high transformation efficiency Escherichia coli (E. coli) HB101. A cDNA library was prepared. 3600 colonies were screened for the light chain and 10000 colonies were screened for the heavy chain.
5. Screening
Therefore gL221A (gL221+D1Q, Q3V, L46R, L47W, see
15.2. Heavy Chain
15.2.1. Extent of the CDRs
The CDR-grafted heavy and light chain genes were co-expressed in COS cells either with one another in various combinations but also with the corresponding murine and chimeric heavy and light chain genes substantially as described above. The resultant antibody products were then assayed in binding and blocking assays with HPB-ALL cells as described above.
The results of the assays for various grafted heavy chains co-expressed with the gL221C light chain are given in
The basic grafted product without any human to murine changes in the variable frameworks, i.e. gL221 co-expressed with gh341 (JA178), and also the �fully grafted� product, having most human to murine changes in the grafted heavy chain framework, i.e. gL221C co-expressed with gh341A (JA185), were assayed for relative binding affinity in a competition assay against murine OKT3 reference standard, using HPB-ALL cells. The assay used was as described above in section 3.3. The results obtained are given in
The binding and blocking assay results indicate the following:
The JA198 and JA207 constructs appear to have the best binding characteristics and similar binding abilities, both substantially the same as the chimeric and fully grafted gH341A products. This indicates that positions 88 and 91 and position 76 are not highly critical for maintaining the OKT3 binding ability; whereas at least some of positions 6, 23, 24, 48, 49, 71, 73 and 78 are more important.
This is borne out by the finding that the JA209 and JA199, although of similar binding ability to one another, are of lower binding ability than the JA198 and JA207 constructs. This indicates the importance of having mouse residues at positions 71, 73 and 78, which are either completely or partially human in the JA199 and JA209 constructs respectively.
Moreover, on comparing the results obtained for the JA205 and JA183 constructs it is seen that there is a decrease in binding going from the JA205 to the JA183 constructs.
This indicates the importance of retaining a mouse residue at position 23, the only position changed between JA205 and JA183.
These and other results lead us to the conclusion that of the 11 mouse framework residues used in the gH341A (JA185) construct, it is important to retain mouse residues at all of positions 6, 23, 24, 48 and 49, and possibly for maximum binding affinity at 71, 73 and 78.
Similar Experiments were carried out to CDR-graft a number of the rodent antibodies including antibodies having specificity for CD4 (OKT4), ICAM-1 (R6-5), TAG72 (B72.3), and TNFα(61E71, 101.4, hTNF1, hTNF2 and hTNF3).
CDR-Grafting of a Murine Anti-CD4 T Cell Receptor Antibody, OKT4A
Anti OKT4A CDR-grafted heavy and light chain genes were prepared, expressed and tested substantially as described above in Example 1 for CDR-grafted OKT3. The CDR grafting of OKT4A is described in detail in Ortho patent application PCT/GB 90 . . . of even date herewith entitled �Humanized Antibodies�. The disclosure of this Ortho patent application PCT/GB 90 . . . is incorporated herein by reference. A number of CDR-grafted OKT4 antibodies have been prepared.
Presently the CDR-grafted OKT4A of choice is the combination of the grafted light chain LCDR2 and the grafted heavy chain HCDR10.
The Light Chain
The human acceptor framework used for the grafted light chains was RE1. The preferred LCDR2 light chain has human to mouse changes at positions 33, 34, 38, 49 and 89 in addition to the structural loop CDRs. Of these changed positions, positions 33, 34 and 89 fall within the preferred extended CDRs of the present invention (positions 33 and 34 in CDR1 and position 89 in CDR3).
The human to murine changes at positions 38 and 49 corresponds to positions at which the amino acid residues are preferably donor murine amino acid residues in accordance with the present invention.
A comparison of the amino acid sequences of the donor murine light chain variable domain and the RE1 human acceptor light chain variable further reveals that the murine and human residues are identical at all of positions 46, 48 and 71 and at all of positions 2, 4, 6, 35, 36, 44, 47, 62, 64�69, 85, 67, 98, 99 and 101 and 102. However the amino acid residue at position 58 in LCDR2 is the human RE1 framework residue not the mouse OKT4 residue as would be preferred in accordance with the present invention.
The Heavy Chain
The human acceptor framework used for the grafted heavy chains was KOL.
The preferred CDR graft HCDR10 heavy chain has human to mouse changes at positions 24, 35, 57, 58, 60, 88 and 91 in addition to the structural loop CDRs.
Of these positions, positions 35 (CDR1) and positions 57, 58 and 60 (CDR2) fall within the preferred extended CDRs of the present invention. Also the human to mouse change at position 24 corresponds to a position at which the amino acid residue is a donor murine residue in accordance with the present invention. Moreover, the human to mouse changes at positions 88 and 91 correspond to positions at which the amino acid residues are optionally donor murine residues.
Moreover, a comparison of the murine OKT4A and human KOL heavy chain variable amino acid sequences reveals that the murine and human residues are identical at all of positions 23, 49, 71, 73 and 78 and at all of positions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
Thus the OKT4A CDR-grafted heavy chain HCDR10 corresponds to a particularly preferred embodiment according to the present invention.
CDR-Grafting of an Anti-Mucin Specific Murine Antibody, B72.3
The cloning of the genes coding for the anti-mucin specific murine monoclonal antibody B72.3 and the preparation of B72.3 mouse-human chimeric antibodies has been described previously (ref. 13 and WO 89/01783). CDR-grafted versions of B72.3 were prepared as follows.
(a) B72.3 Light Chain
Comparison of the murine B72.3 and REI light chain amino acid sequences reveals that the residues are identical at positions 46, 58 and 71 but are different at position 48. Thus changing the human residue to the donor mouse residue at position 48 may further improve the binding characteristics of the CDR-grafted light chain, (B72.3 gL) in accordance with the present invention.
(b) B72.3 Heavy Chain
i. Choice of Framework
ii. Results with Grafted Heavy Chain Genes
iii. Framework Changes in B72.3 gH Gene
iv. Other Framework Changes
v. Other
CDR-Grafting of a Murine Anti-ICAM-1 Monoclonal Antibody
A murine antibody, R6-5-D6 (BP 0314863) having specificity for Intercellular Adhesion Molecule 1 (ICAM-1) was CDR-grafted substantially as described above in previous examples. This work is described in greater detail in co-pending application, British Patent Application No. 9009549.8, the disclosure of which is incorporated herein by reference.
The human EU framework was used as the acceptor framework for both heavy and light chains. The CDR-grafted antibody currently of choice is provided by co-expression of grafted light chain gL221A and grafted heavy chain gH341D which has a binding affinity for ICAM 1 of about 75% of that of the corresponding mouse-human chimeric antibody.
Light Chain
gL221A has murine CDRs at positions 24�34 (CDR1), 50�56 (CDR2) and 89�97 (CDR3). In addition several framework residues are also the murine amino acid. These residues were chosen after consideration of the possible contribution of these residues to domain packing and stability of the conformation of the antigen binding region. The residues which have been retained as mouse are at positions 2, 3, 48 (?), 60, 84, 85 and 87. Comparison of the murine anti-ICAM 1 and human EU light chain amino acid sequences reveals that the murine and human residues are identical at positions 46, 58 and 71.
Heavy Chain
gH341D has murine CDRs at positions 26�35 (CDR1), 50�56 (CDR2) and 94�100B (CDR3). In addition murine residues were used in gH341D at positions 24, 48, 69, 71, 73, 80, 88 and 91. Comparison of the murine anti-ICAM 1 and human EU heavy chain amino acid sequences are identical at positions 23, 49 and 78.
CDR-Grafting of Murine Anti-TNFa Antibodies
A number of murine anti-TNFa monoclonal antibodies were CDR-grafted substantially as described above in previous examples. These antibodies include the murine monoclonal antibodies designated 61 E71, hTNF1, hTNF3 and 101.4 A brief summary of the CDR-grafting of each of these antibodies is given below.
A similar analysis as described above (Example 1, Section 12.1.) was done for 61E71 and for the heavy chain 10 residues were identified at 23, 24, 48, 49, 68, 69, 71, 73, 75 and 88 as residues to potentially retain as murine. The human frameworks chosen for CDR-grafting of this antibody, and the hTNF3 and 101.4 antibodies were RE1 for the light chain and KOL for the heavy chain.
Three genes were built, the first of which contained 23, 24, 48, 49, 71 and 73 [gH341(6)] as murine residues. The second gene also had 75 and 88 as murine residues [gH341(8)] while the third gene additionally had 68, 69, 75 and 88 as murine residues [gH341(10)]. Each was co-expressed with gL221, the minimum grafted light chain (CDRs only). The gL221/gH341(6) and gL221/gH341(8) antibodies both bound as well to TNF as murine 61E71. The gL221/gH341(10) antibody did not express and this combination was not taken further.
Subsequently the gL221/gH341(6) antibody was assessed in an L929 cell competition assay in which the antibody competes against the TNF receptor on L929 cells for binding to TNF in solution. In this assay the gL221/gH341(6) antibody was approximately 10% as active as murine 61E71.
hTNF1 is a monoclonal antibody which recognises an epitope on human TNF-α. The EU human framework was used for CDR-grafting of both the heavy and light variable domains.
Heavy Chain
In the CDR-grafted heavy chain (ghTNF1) mouse CDRs were used at positions 26�35 (CDR1), 50�65 (CDR2) and 95�102 (CDR3). Mouse residues were also used in the frameworks at positions 48, 67, 69, 71, 73, 76, 89, 91, 94 and 108. Comparison of the TNF1 mouse and EU human heavy chain residues reveals that these are identical at positions 23, 24, 29 and 78.
Light Chain
In the CDR-grafted light chain (gLhTNF1) mouse CDRs wre used at positions 24�34 (CDR1), 50�56 (CDR2) and 89�97 (CDR3). In addition mouse residues were used in the frameworks at positions 3, 42, 48, 49, 83, 106 and 108. Comparison of the hTNF1 mouse and EU human light chain residues reveals that these are identical at positions 46, 58 and 71.
The grafted hTNF1 heavy chain was co-expressed with the chimeric light chain and the binding ability of the product compared with that of the chimeric light chain/chimeric heavy chain product in a TNF binding assay. The grafted heavy chain product appeared to have binding ability for TNF slightly better than the fully chimeric product.
Similarly, a grafted heavy chain/grafted light chain product was co-expressed and compared with the fully chimeric product and found to have closely similar binding properties to the latter product.
hTNF3 recognises an epitope on human TNF-α. The sequence of hTNF3 shows only 21 differences compared to 61E71 in the light and heavy chain variable regions, 10 in the light chain (2 in the CDRs at positions 50, 96 and 8 in the framework at 1, 19, 40, 45, 46, 76, 103 and 106) and 11 in the heavy chain (3 in the CDR regions at positions 52, 60 and 95 and 8 in the framework at 1, 10, 38, 40, 67, 73, 87 and 105). The light and heavy chains of the 61E71 and hTNF3 chimeric antibodies can be exchanged without loss of activity in the direct binding assay. However 61E71 is an order of magnitude less able to compete with the OFF receptor on L929 cells for TNF-α compared to hTNF3. Based on the 61E71 CDR grafting data gL221 and gH341(+23, 24, 48, 49 71 and 73 as mouse) genes have been built for hTNF3 and tested and the resultant grafted antibody binds well to TNF-α, but competes very poorly in the L929 assay. It is possible that in this case also the framework residues identified for OKT3 programme may improve the competitive binding ability of this antibody.
101.4 is a further murine monoclonal antibody able to recognise human TNF-α. The heavy chain of this antibody shows good homology to KOL and so the CDR-grafting has been based on RE1 for the light chain and KOL for the heavy chain. Several grafted heavy chain genes have been constructed with conservative choices for the CDR's (gH341) and which have one or a small number of non-CDR residues at positions 73, 78 or 77�79 inclusive, as the mouse amino acids. These have been co-expressed with cL or gL221. In all cases binding to TNF equivalent to the chimeric antibody is seen and when co-expressed with cL the resultant antibodies are able to compete well in the L929 assay. However, with gL221 the resultant antibodies are at least an order of magnitude less able to compete for TNF against the TNF receptor on L929 cells.
Mouse residues at other positions in the heavy chain, for example, at 23 and 24 together or at 76 have been demonstrated to provide no improvement to the competitive ability of the grafted antibody in the L929 assay.
A number of other antibodies including antibodies having specificity for interleukins e.g. IL1 and cancer markers such as carcinoembryonic antigen (CEA) e.g. the monoclonal antibody A5B7 (ref. 21), have been successfully CDR-grafted according to the present invention.
It will be appreciated that the foregoing examples are given by way of illustration only and are not intended to limit the scope of the claimed invention. Changes and modifications may be made to the methods described whilst still falling within the spirit and scope of the invention.
Patent Citations
Non-Patent Citations
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The Observation Deck
A blog of opinions on local, state and national issues
Posts filtered on Category
What to talk about if you're out of ideas
In the absence of genuinely good ideas about how to turbocharge the economy, Republicans have dangerously decided the best course forward is to focus on immigration, says Juan Williams in Fox News. Will it work? "Stay tuned," he says. [Read More]
Border starting to look like a war zone
With drones overhead, massive imposing borders and heavily armed militaries pursuing an enemy that easily blends into the civilian population, the drug war on the Mexican border is increasingly more like Afghanistan, says James Poulos in Foreign Policy. [Read More]
'Illegal' as a noun is nothing but a slur
The term "illegal," used by Republicans as a noun, is a slur used against Mexicans, says Peter Beinart in The Daily Beast. As proof, note that they don't use the term for others who break the law, including those who illegally hire undocumented workers, he says. [Read More]
Oh no, the illegals are leaving!
In a satirical look at United States immigration policy, Gawker presents a highlight reel of a South Park episode that imagines what immigration at the Mexican border might look like when economic conditions deteriorate to the point that immigrants decide to go back home. [Read More]
Border security a giant sucking sound
The southern border of the United States is not the violent no-man's land that Rick Perry and other politicians portray it to be, says Veronica Escobar in The New York Times. Security measures are wasting resources that cry for use elsewhere, she says. [Read More]
Fair play, or out-of-control spin?
Elections are all about controlling the message, and part of that comes in defining your opponent. Doing so often means spinning what the opponent says, whether you're misrepresenting it or not. Two cases in point this week from the Republican CNN/Tea Party debate Monday. In one, the liberal ThinkProgress was among those that took note […] [Read More]
Undocumented but not unskilled
It's easy to conclude that undocumented equals unskilled, says Megan McArdle in The Atlantic. Undocumented immigrants do jobs that most Americans are unwilling to do, and also have skills in jobs like agricultural harvesting that most of us do not have. [Read More]
An all-American quiz (updated with poll)
So you're a proud American citizen, eh? Well, for the July 4th weekend, we've picked out 20 questions, taken from the official U.S. citizenship test. See how much you know about government, American history, civics and geography. Feel free to leave your score in a comment. Normally, prospective citizens are given 10 questions, chosen by […] [Read More]
Dream act better for kids, America
The Dream Act is an essential part of immigration reform, so says U.S. secretary of Education Arne Duncan in USA Today. The Dream Act will allow the children of undocumented immigrants a chance to succeed and contribute to a better America, Duncan says. [Read More]
An immigration plan. Mr. Obama?
Our opinion: Governor Cuomo and leaders of two other states have grave doubts about a White House plan to deport criminals. Where's the focus of keeping families together, and finding a way for illegal immigrants to become legal? Governor Cuomo wants no part of a federal effort intended to deport dangerous felons whose crimes include entering the […] [Read More]
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subset: commoncrawl


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OP Member
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Join Date:Joined: Dec 2012
and there are other similar charging problems i get to understand that are stemmed from this problem
so if u have any charging problems i suggest u try it before u send it off to the repair shop and spend unnecessary money
first thing you should always do is to try another charger or usb cable... preferable one that has a real tight fit
ok now to the solution.. incase you didnt know when you carry around your phone in your pocket some loose fabric gets into the charging port which causes alot of issues.
it can cause:
loose charger
slow charging
pc non recognition problems
no charging at all
etc. basically anything that has to do with the charging port
all u need to do is get like a needle or a small pin or something of that nature and clean out that fabric out of the port... and if possible blow it out after to make sure that any loose fabric the needle or pin loosened is removed
be careful when doing this ! dont jam down in to it..just gently move it around in there and try not to damage anything. TAKE EXTREME CARE. BE WARNED.


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|ESPN: SportsNation
Thanks for coming by.. it's been another interesting week in the CF off season. Let's get to it.
Any word why Willy Korn chose to transfer from Marshall?
I'm guessing because he doesn't want to play DB and still wants to be a QB. From what I'd heard out of Marshall, Korn did very well at safety and the coaching staff loved him. but then they didn't hear from him for two days and he was gone.
With the addition of Nevada and Fresno St and Boise St, can you see the MTN West getting an Auto-bid to the BCS
Not if BYU leaves in addition to Utah. Fresno and Nevada haven't played at that level. You've strengthened the middle but not the top of the league.
And you've turned the WAC into something less than the Sun Belt.
What's it going to take to get Jake Locker or Andrew Luck in the Heisman conversation early in the season?
If Locker leads Washington to wins in three of their first four games, he'll be in very good shape because they have BYU, Neb and at USC plus Syracuse.. there's some buzz about him right now. Luck needs a big win at ND and then at Oregon. I don't think either guy can win it unless their team wins at least nine or 10 and Luck may need Stanford to win one more game because he's younger.
Why is it that Masoli is eligible to play this year while players that transfer even if they dont play a down have to sit out a year? Doesnt sound like a fair rule
Because he has already graduated and there's an out rule that if you transfer to a school that has a graduate program that the school you're at doesn't, you can go play without penalty.
Does Wisconsin have a chance to play in the Rose Bowl this season?
Yes, a pretty good shot. They have a great O-line, capable QB and a big-time RB. And they get OSU at home.
Who would you say ends up having the better year? The Vols or the Wolverines?
Wolverines. They're much better on the O-line.
Any kind words for Arizona State? Think they might be a little underrated? That is one heckuva defense led by Lawrence Guy, Vontaze Burfict and Omar Bolden!
They have a cool logo, a funny head coach and like you said some big-time young defensive players. The irony is the offense is the sticking point now and might be the thing that holds Erickson from getting another season there.
Best WR/TE combo in the nation...Floyd/Rudolph?
Yes, although Arkansas has a great 1-2 in Childs and DJ Williams.
Will I get fired after this season?
I wrote about this in today's mailbag... yes, I'm afraid so. Tough non-conference start to get going.
Where do you see UGA finishing in the east this year?
Second behind UF although if their D doesn't get a lot better I could see third behind Carolina.
Of Miami's ACC games, which two do you think will be the Canes' hardest?
Even though both are home I'll go FSU and VT.
USC is where in 5 years?
Good question.. I think they'll be good this year. Maybe 11-2.. then 9-3/10-2 in 2011. I think the first year they're eligible for postseason, they could make a BCS title run assuming Barkley stays for his senior year. They have a lot of big-time freshmen and sophs. Depth will obviously be and issue but I think they'll be really good. They skill talent there now is really impressive. This class he just signed looks special. It's 2013, where I think the numbers catch up to them and they could be looking at a 7-5 type of year.
Bruce, Be realistic...is the defense of Ark strong enough to win the SEC?
I don't think so. Not to overtake Bama in the West.
Just for kicks: What program(s) do you think will surprise everyone in this season? (Great pieces on ND, by the way. Finally, a sports writer who gets what the school is all about. Kelly's doing wonders for the program, too. He gets it.)
Thanks..Am on record saying Wisc. will be a lot better than pollsters think. I'm intrigued by Texas A&M as well. Off the radar, I'll say Houston and Middle Tennessee.
Is bill snyder slowly building KSU back into a big 12 power? Brown brothers, harper, good recruits coming in.
At his age, it'd better not be slowly. Not too sure how good the Brown brothers will be. I'd heard Arthur runs well but wasn't the instictive kind of LB that you'd hope for... putting these pieces together at K-State and finding the JC success Snyder had 15 years ago is a tough recipe to repeat.
Who do you like in the Pac-10 this year?
Of the 9 eligible teams for the title, I'll go with Oregon. Lots of experience back and a super fast backfield.
Any idea where elite TE recruit Nick O'Leary of Dwyer HS (Fla.) is leaning?
Not sure yet. Been meaning to write about him this off-season...I was so impressed when I saw him play in the state title game last winter. The guy can do everything on the football field. Tough, Agile, blocks, great hands. Jack Nicklaus' grandson is the real deal.
Which teams pre-season poll ranking will be the biggest "mistake"? You can go in either direction with this one.
I was surprised that Penn State was at #14 with a new QB and having to replace half the D to go with three tough road games. Not surprised they were ranked, but I thought #14 was too high.
Biggest reason Virginia Tech won't beat Boise State?
Well, I think they will, but since you asked.... because Boise State is fantastic at keeping defenses off balance with all of that motion and different looks.
The best thing about your job is...
I cover a sport I truly love all the time. If I won the lottery, I'd sill try and keep up with college football. I love the sport as much as I did 10 years ago, if not more..
How will McNair's appeal to the NCAA affect USC's appeal to have their sanctions reduced?
It seems almost impossible to win those NCAA appeals. If you examine the McNair case, it raises some valid questions about the NCAA investigation and he is the only direct link the NCAA established in knowing what was going on with Reggie Bush.... I also was surprised after I'd initially heard the punishments then read the next day from the NCAA what they flagged USC for. I had figured it must've been more football players caught than Bush... Still, I don't think USC fans should be optimistic about winning the appeal to get a year of post season back or some scholarships. It's the NCAA. They basically do what they want.
Do you think it was a huge mistake for USU not to go to the MWC?
Yes, I do.
What does Miami have to do to beat Ohio State at The Shoe on September 11?
Protect Jacory Harris and have the UM D-line stop OSU from running the ball.
Mark May's got folks aTwitter by saying Denard Robinson has been named Michigan's starting QB against UConn. What's your take on the UM quarterback situation. Is everything rosy there? (Not Rose Bowly, of course.)
I think Robinson's explosiveness as a runner is something that UM hasn't really had since Rodriguez has been there. With his more experience in the system and maturity, he will play faster this year, RichRod told me. That ability will get defenses on their heels more. I do think you'll see Forcier play a lot too, but my hunch is Robinson will be tough to overtake.
Who is the best player, regardless of position, in the country? The best coach?
Saban or Urban Meyer... I'll say either LSU CB Patrick Peterson or UNC DE Robert Quinn.
What do you make of BYU jumping to independent status?
Also covered this in more detail in the blog today.. I think it's a big mistake.
Who are your All-Time Favorite College Running Backs? Thanks
Tough to pick just one.. Earl Campbell was one of the first CF players I ever really noticed and I loved him in the NFL... Others that came to mind: Barry Sanders, Maurice Jones-Drew, Greg Jones, Dexter McCluster.
Hey Bruce! How do you think the UM transfer Marve will do at Purdue? Will they be good enough to be bowl eligible? Thanks
Yeah, everyone around Purdue is so high on him. I saw him a lot in high school. He has a great arm and is very athletic and tough. I think he'll get them into a bowl this year and into the top 25 in 2011.
Is Autzen the loudest stadium west of the Mississippi?
My geography is shaky, but I'll say it's the loudest from the Big 12 and west of that.
Where is said blog?
Will the Cal Bears see Rose at the end of the Year
I don't think they have the QB play to get to that level. The non-conference is very manageable and I think they should be pretty good, but my hunch on them is 7-5. I don't see more than 8-4 unless Kevin Riley makes huge improvement, and I'm skeptical at this point.
Jones-Drew? Greg Jones?? McCluster?? And you won't say Reggie Bush? Are you kidding me? Your bias against USC is very obvious.... Not because of this.. but because most of your articles. Reggie is probably the most exciting CFB RB of all time...
I was asked who my favorite RBs were... there were certain things I really liked about those guys. Some style, some personality. Sorry. Anyhow, we all get accused of being biased against every school at some point.
Can Christian Ponder win the heisman this year?
He is good enough and he has a very good O-line. Losing WR Jarmon Fortson hurts his cause I think, but if they can win at OU, which no one has done in a very long time, he'll be in the hunt.
Do you see more conference expansion happening?
I do. I think the Big Ten will go east at some point in the near future and the Big 12 looks pretty shaky.
Any chance of josh nesbitt being drafted to play something other than qb?
Yeah, I could see that as a safety. I know a guy who was on the GT staff and said Nesbitt was the toughest guy on the team. He's athletic and smart. He might be a really good defensive player.
Hi Bruce, I think LSU is going to loose to Alabama and Florida and maybe Ark. to. If they loose them games plus one more ( maybe UNC) will LES be gone? (to Michigan maybe)
He loses those plus UNC and I think LSU will look for a new head coach. Pressure there is a very high. Expectations at LSU skyrocketed in the last decade. I do think the Tigers will have a better year than that though.
This year is termed as a "rebuilding" year for tennessee, how much rebuilding is being done there? I think Dooley will have success given enough time, which I think he'll get because he's a safe coach(see Lane Kiffin debacle)
I'm not sure this will work out for them. He's digging out of a hole and it might get worse before it gets much better. Not sure they will be that patient there.
What do you think of Spurrier lobbing some verbal grenades at Tennessee?
Spurrier being Spurrier. He actually borrowed a page from Lane Kiffin. Spurrier has his own little mess at So Carolina and he took a step back and zinged UT--and UNC.
Why would Les be fired from a 2nd tier job (below the truly elite ones) and go to one of the all time great programs? lol
I don't follow.. are you saying LSU is a second-tier job?
Does Pitt finally get the Big East BCS bid this year? Also what about Lewis' chances for Heisman
I think so and he has a shot. He's very good. Curious to see how he does against Miami in a few weeks and then at ND. He'll be on a big stage in those two if he can put up big #s.
How many games does RRod have to win to keep his job?
I'll say 8.. Seven might do it if he knocks off Ohio State or even Wisconsin or Iowa. Seven could be dicey if he opens 5-1 and then they backslide to 2-4 down the stretch.
Are there any games you are particularly looking forward to this season?
Seriously, all of them. Can't wait. Going to LSU-UNC first weekend and then Boise-VT.
I take it back. Wikipedia fixed my opinion. Still, LSU -> Michigan is an UPGRADE, even if not as much as one as I suggested. Peace.
If Wikipedia didn't, I could post some of the things other folks have sent in to make the Tigers case.
Do you think Ole Miss with Masoli will be better than Ole Miss with Snead in 2008?
I don't. That Ole Miss team had Mike Wallace, Dexter and Shay Hodge. Those are three big time weapons. Masoli has Lionel Breaux, Markeith Summers and JEssie Grandy who are nice players, but not at that level.
Bruce,One more LSU question. You are truly one of the few national commentators that think LSU will have a good season. Why?
I think their QB play for the first time in a few years will be solid. I also think if they get past UNC in the opener, and UNC is clearly coping with a lot of turmoil, they have a good chance to get some momentum going before they head to Gainesville in Wk 6.
How much anticipation do you have for the second week of the season with soooo many big time non-conference match-ups? How are you going to be watching?
My plan is to drive from DC for the Boise-VT game and stay on the east coast and head to Columbus for Miami-OSU.
Three more questions...
LSU always gets ranked so high when they beat nobody. They lose every year to FL and Bama, so they get a pass for that and remain in the top 25. I cant stand it. They dont play anybody!
I just looked this up. LSU has played 16 teams that were ranked in the top 12 over the past four seasons. That's not bad.
played, not beaten
They beat nine of those 16.
Houston and SMU to the MWC?
Wouldn't shock me at this point.
You hear anything re: who will be the QB at Penn State?
Hearing young Robert Bolden, the true freshman, is making it very interesting. Whoever starts is going to be inexperienced. I saw Bolden last summer at Elite 11 and was really impressed. He has good feet and really puts a lot of zip on the ball. Figured he'd be a good one for them by 2012. Maybe he's ahead of schedule.
Thanks for all of the questions. If I didn't get to yours, send it to me via Twitter at BFeldmanESPN or into my blog.


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Dorothy I. Brockhaus, 87, a resident of Churubusco and widow of Edward Brockhaus, Jr., passed away Friday, Dec. 30, 2011, at 9:55 p.m. at Parkview Hospital in Fort Wayne.
She had been a patient there for the past month.
Mrs. Brockhaus was born Jan. 26, 1924, in Defiance, Ohio, a daughter of John and Daisy (Boterf) Lipp.
She spent her formative years in that area and graduated from Sherwood Delaware High School in 1941.
Strong winds will accompany the coldest air of the season so far.
Hold on to your trash cans and recycling bins, Mother Nature and Old Man Winter are teaming up for a special treat. Spent Christmas trees put out at the curb may find their way into the middle of the road, thanks to gusty winds.
As is often the case with some of the coldest outbreaks of the winter, strong winds will add to the shock of the dramatic change in weather during the first few days of the new year.
Wind gusts of 30, 40 and even 50 mph will blast from the Midwest to the Atlantic Seaboard over the next couple of days.
HUNTERTOWN — In last year's sectional title game against Wawasee, Columbia City's boys basketball team fell to a 10-0 deficit in the first quarter but came back to win the title 67-66
The Eagles didn't have the same luck in Friday's holiday shoot-out championship game against Homestead.
The Spartans jumped out to a 16-0 lead in the first quarter and Columbia City never recovered, picking up its second loss of the season, 63-45.
"It was disappointing to come out to a team that we knew was going to be good and not put our best foot forward," Columbia City Head Coach Chris Benedict said.
PERU — Guaranteed to at least match the girls' second place finish, Whitko boys basketball took the court Friday in the final game of Peru's Holiday Tournament.
The Wildcats faced, and beat, the LaVille Lancers by a final of 64-55.
The half ended with a difference of just one point, 26-25 in favor of LaVille. At the break, Logan Irwin had 13 of his team leading 34 points.
Though keeping the score close, the Wildcats were just 7-for-29 in the first half (.241).
In the third quarter, following the fifth tie of the game (29), the Lancers scored 11 of the next 14 points.
CHURUBUSCO — Micheal E. Sherman, 72, a resident of Urbana, Ohio and formerly of Churubusco, passed away at 3:23 p.m. on Thursday, Dec. 29, 2011, at his home.
He was born on May 26, 1939, in Arcola, a son of Virgil C. and Edna (Butler) Sherman.
His early years were spent in the Arcola area until moving to Noble County near Merriam with his parents.
He graduated from Wolf Lake High School.
Brian Lee Baker, 53, departed this life on Tuesday, Dec. 13, 2011 at his home in Fort Wayne.
Born March 29, 1958, to Paul and Jo Ann Baker in Fort Wayne, Brian graduated from Whitko High School.
He worked at Dana Corp. for 30 years, retiring in 2007. Brian enjoyed all sports.
Dorothy J. Mittendorf, 86, a resident of Huntertown, died at 2:26 p.m. on Wednesday, Dec. 28, 2011, at Miller's Merry Manor in Garrett.
She was born Dec. 17, 1925, in Summitville, a daughter of Stephen and Myrtle (Poindexter) Ellsworth.
On Feb. 1, 1947, she was married to Ralph Mittendorf, Jr.
Survivors include her husband, Ralph, Jr.; her son, David L. Mittendorf of Huntertown; a brother, Jerome J. Ellsworth of Dearborn, Mich.; a sister, Barbara Weber of Rock Island, Ill.; four grandchildren and seven great-grandchildren.
Howard S. Strader, 82, of Fort Wayne, passed away at 5:58 a.m. Thursday, Dec. 29, 2011, at Visiting Nurse and Hospice Home, Fort Wayne.
Born Oct. 13, 1929, in Akron, Ohio, he was the son of William and Mildred (McPhillen) Strader.
He was a resident of Allen County until moving to Whitley County in 1951.
He attended St. Joe Township Schools, Allen County.
COLUMBIA CITY — The Columbia City Electric Department put the finishing touch on a nearly two-year project Friday.
The department, doing most of the work in its spare time, has successfully changed out over 4,500 electric meters, the device that measures the amount of electric energy consumed by a residence, business, or electronically powered device.
Two years ago the city was contacted to gauge interest in purchasing an over-stock of meters at a discount price.
"We got them for a very inexpensive price," said Electric Department Superintendent Larry Whetstone.
PERU — Both Whitko's boys and girls basketball teams are advancing to the championship games at the Peru tournament tonight.
The girls won the tournament last season and are looking to do the same this year after a 69-48 win over LaVille Thursday night.
The Lady Wildcats (5-6) face host Peru (4-7) tonight in the championship game at 6 p.m.
The boys picked up their second win of the season Thursday against Peru, 66-50.
Logan Irwin had six 3-pointers in his 34 points.


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A guide to NC's proposed fracking rules
Areas covered by the proposed rules: Geophysical surveys, drilling units, well spacing, permitting, financial assurance, well site construction, well construction, chemical disclosure, baseline water testing, water sourcing, waste disposal, site reclamation, and operation and production.
Areas not covered by the proposed rules: Compulsory pooling, air quality, regulatory fees, road use, stormwater control, waste disposal, fees and taxes.
Samples of the Mining and Energy Commission 's Proposed Rules
Emergency response planning: Permit holder must identify types and amounts of chemicals, fluids and wastes present at a production facility. Permit holder must submit emergency plans for chemical spills, fires and explosions.
Setback distances: Gas wells, tanks and pits must be at least 650 feet from homes, buildings and drinking water wells; at least 200 feet from rivers, ponds and other waterways; at least 100 feet from roads, utility/rail rights-of-way and intermittent streams. Exceptions to setbacks require a variance granted from the Mining and Energy Commission.
Prohibited substances: The following substances are prohibited from being injected into the ground during fracking or disposal: diesel, fuel oil, kerosene and crude oil.
Well maintenance: Drilling sites must have a functioning blowout preventer on site during maintenance, remediation and fracking operations.
Well closure: Upon abandonment of wells, well bores will not be plugged with trash or other non-drillable material.
Chemical disclosure: Permit holder will disclose online any fracking additives and hazardous chemicals, including chemical formulas, quantities and concentrations, to local emergency responders and to the N.C. Department of Environment and Natural Resources.
Trade secrets: Additives and chemicals can be shielded from disclosure, but trade secret exemptions require approval from the N.C. Mining and Energy Commission. The confidential information must be provided upon request to health care providers, fire chiefs and state agencies if required to perform their functions.
Baseline water testing: All water supplies within half-mile of a proposed gas wellhead will be tested before drilling and up to five times after drilling. The test samples will be analyzed for arsenic, barium, radium, benzene and diesel, among other substances. The testing will be conducted by an independent lab, paid for by the permit holder, and made available to the public.
Water storage: Wastewater and flowback water can be stored in open air pits, open tanks or closed tanks. If an exposed pit is used, the pit must be double-layered with synthetic liners and equipped with leakage monitors between the liners. The permit holder must inspect pits and open tanks after half-inch of rain falls within a 24-hour period.
Water supply contamination: The permit holder will replace a property owner's water supply if lab results show the water supply was contaminated by the permit holder's activities.
Water sourcing: Permit holder will investigate alternatives to using fresh water, including flowback and produced water, and provide a reason for not using the alternatives if they are rejected.
Waste management: Permit holder must submit a management plan for storing and handling wastewater and solid wastes from the entire exploration process including final disposal.
Proposals and recommendations (from study groups, advocacy organizations and N.C. Attorney General)
Compulsory pooling: Should be allowed only when 90 percent of landowners have signed fracking leases. (Under current state law, forced pooling could happen if just one landowner in a drilling unit signed a fracking lease.)
Blowout mitigation: Permit holder should be required to have a hazard mitigation contract with a company specializing in well disaster mitigation.
Chemical disclosure: Everything should be disclosed, no trade secret exemptions.
Posting violations: All violations related to hydraulic fracturing in the state should be posted online.
Air emissions: North Carolina should require monitoring and control of air pollution emissions caused by hydraulic fracturing activities, including methane leaks and diesel fumes from wells, generators, trucks and heavy equipment used in fracking operations.
Provisions that already exist in North Carolina law
Presumptive liability: If water supplies are contaminated, it is presumed the gas operator is responsible for the water contamination within half-mile of a gas wellhead, unless the operator can prove otherwise.
Compensation for damages: A gas operator must compensate property owner for damages caused to water supply, personal property, livestock, crops or timber.
Site reclamation: A gas operator is required to restore all surface areas within two years of completing operations.
Bonding: The operator must provide a bond to cover $1 million in environmental damage.
Landmen: A landman is an agent who solicits leases for rights to drill and explore for natural resources from property owners. Landmen must register with the state before soliciting leases from landowners.
Leases: When fracking leases are offered, they must include a publication outlining landowner rights. The leases must include a conspicuous disclosure that landowners should obtain approval from mortgage lenders before signing a fracking lease. Either party can cancel a lease within seven business days of signing it.
Mineral rights: Sellers of real estate must disclose to potential buyers if oil and gas rights are "severed" and would not transfer to the buyer of the surface property when the land is bought or sold.
Local laws: Local governments will not pass ordinances that prohibit fracking or have the effect of preventing shale gas exploration.
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The Top 10 In Action Sports
01Welcome to Qasr Al-Farid: The Rock With A Door That Will Rock Your World
0218 Things That Will Give Anyone Who Did Duke Of Edinburgh A Serious Flashback
0316 Things That Will Give 90s Skater Kids A Serious Flashback
04This 10 Year Old Kid Just Nailed A Dub Cork 1080... On His Second Attempt
05The 10 Most Spectacular Road Trips on Earth
06Water Slides, Plane Wings and Danny MacAskill: This is Road Cycling Like Never Before...
0724 Reasons Why Snowboarding Was Better In The Early Noughties
08Could Scientists Be About to Build a Terminator? They're Closer Than You Think…
09Win a Holiday for 2 in Chamonix with Three #holidayspam
1017 Holiday Beer Photos That Every Skier Takes to Make Their Friends Jealous
The Backwards Teaser
A Snowboarding video by keltron - 5,224 views
Fearing "The B" could be possessed, it was played backwards to check for hidden messages. It looked cool, thus the backwards teaser. Burton's latest movie "The B" will hit stores and iTunes, ghost free, this fall.
The B is a major snowboarding motion picture showcasing the riding of the entire Burton team.
What Do You Think?
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Illinois African-American Clergy Join the Fight for Marriage Equality
October 18, 2013 by Lynne Bowman, Regional Field Director
Yesterday morning, a dozen African American clergy gathered at the Chicago Temple to publicly give voice to the support that exists among African American people of faith for marriage equality. Led by the Rev. Benjamin Reynolds, HRC employee and Director of Faith Organizing for the Illinois Unites for Marriage campaign, the pastors spoke of the tenets of freedom and fairness that are upheld in Senate Bill 10, the Religious Freedom and Marriage Fairness Act. And they called on the legislature to pass the bill when they return to session next week.
The clergy were joined by Rev. Delman Coates, a leader in the successful 2012 marriage campaign in Maryland. Rev Coates spoke of the delineation between civil and religious marriage and pointed to the strong support of civil marriage equality among people of faith in both Maryland and Illinois.
With the Illinois Veto Session set to begin in less than five days, the announcement from clergy couldn't come at a better time. As legislators prepare to make their final decision about where they will stand when the vote is called, we hope they will listen to the majority of Illinoisans and to this gathering of clergy and vote to protect the freedom of all people while recognizing the fairness that must be inherent in our civil laws.
Video of the press conference can be found here.
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How Long Do I Wait To Get Tested After Swallowed Blood
Here's a taste of what TheBody.com has to offer on this topic:
Get the Facts
Systemic Candidiasis...they only have contact with the mouth for a short time. However, they may be the best choice for someone with a very sore or dry mouth. Rinses are held in the mouth for as long as possible and should be swilled around and then swallowed. They are used at least four times a day and continued for a few days after the symptoms have gone. The most widely used rinse is nystatin (Mycostatin Oral Suspension). Lozenges For oral symptoms, one or two...
Ask the Experts
BJ and swallowedHello Dr Bob!I'm 22 YO and I'm writing from Venezuela... I've been reading this forum for more than one year.My question is... Through this year... I performed oral sex to 3 friends... they cummed in my mouth and I swallowed on the three separated...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...that would create abrasions or cuts in your mouth before performing oral sex. Use mouthwash or a breath mint instead. Avoid swallowing pre-cum, semen, or vaginal fluids. Use latex condoms for oral sex on a man (try the unlubricated, flavored... Read more »
One Night Stand From Your Past... Once AgainSorry, doc. I'm a nervous wreck now. I took comfort in your post until I came down with the exact same symptoms of ars. At around 11 days or so I started coming down with an itchy throat kind of hurt. The next day I had a rash on my left chest. I...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...fingered me for a couple of minutes. Told him to stop because it wasn't feeling good. After this I look at his finger and he had a bloody cuticle/hangnail. It looked dry or "dryish". What I'm fearing is what if it bled again while inside of me.... Read more »
The accuracy of OraQuick/Orasure? Cant even mini woo hoo?? (RELIABILITY OF ORAQUICK ORAL TESTS, 2009)Hi Dr Bob,I just did a rapid OraQuick or Orasure, not sure. The doc swabbed my lower and upper gums, not sure if they swabbed my cheeks, but he swabbed it very quick (I am sure not more than 1-2min) and I am not sure if it more than 1 min and I have...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...2006I have two situations..please help.1) Oral sex with male (I am female) in June of last year. Last 1 minute, ejaculation, swallowed immediately no oral health problems. Have neven gotten the flu or anything close to it, however 7 weeks later,... Read more »
Hepatitis B / Antibiotics and HIV Test (ANTIBIOTICS AND HIV TESTING, 2009)I currently have Hepatitis B and have just finished taking azithromycin for a cough.I just took a Home Access Hiv test (6 weeks after potential exposure) and was wondering if either the Hep B or antibiotics would affect the negative result.Thank you.
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...a unique question about Ora-quick and anti-biotics !!! In april 2003 i had oral sex with other man. I am bi-sexual male. he swallowed me and i did oral on him for about a minute. i guess he might be pre-cumming but not so sure . He did not... Read more »
HIV Transmission...HIV-negative, and avoid giving it to someone else if you are HIV positive. HIV is spread through the following body fluids: Blood (including menstrual blood) Semen and other male sexual fluids ("pre-cum") Vaginal fluids Breast milk ... Read more »
HIV and Safer Sex...you perform oral sex without a condom, finish up with your hand, or spit semen out and rinse with a dental wash rather than swallowing. Dental dams are squares made from latex. Put some water- or silicone-based lube on one side of the dental dam... Read more »
Understanding HIV-Related Lab Tests I: Complete Blood Count and Blood Chemistry... Lab Tests Are Important Tools The Basics Complete Blood Count (CBC) Blood Chemistry (Chemistry or Chem Screens) The Bottom Line Lab Tests Are Important Tools Having regular Read more »
Fungal Infections...Usually occurs at very low CD4 cell counts (less than 100) An AIDS-defining opportunistic infection Major symptom is painful swallowing (dysphagia) Often diagnosed via gastrointestinal endoscopy Possible in anyone who has thrush and a... Read more »
Connect With Others
I just recently tested Positive
Posted by Dakota, 4 RepliesI am so depressed and Stressed I am just at my end. I feel so alone and no one to talk too Read more »
What was I thinking????
Posted by Anonymous, 3 Replies...that careless about another individual? I told my GF what happened when I got back and she is still here, not sure for how long once I get the results I am expecting. worst of all, I just had to pass on a new job in pharmaceutical manufaturing... Read more »
HIV Antibody Testing
Posted by sunkyst, 3 Replies...Does anyone know why HIV antibody tests are done, rather than just directly checking for the HIV virus in the bloodstream? In HIV+ people, their blood can be checked for viral loads, why can't they do this when doing a HIV test, rather than Read more »
I don't know what to think anymore.
Posted by Anonymous, 7 RepliesI recently put up a post explaining that 24 hrs after giving my bf a blow job I was sick with tonsilitis. I explained that I didn't swallow any cum and very little pre cum. I was satisfied with the response that I probably didn't have HIV, until... Read more »
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Actor Val Kilmer on Wednesday apologized for comments attributed to him that many residents have considered derogatory toward Hispanics and veterans.
Under a heavy police presence at the old courthouse, the San Miguel County Commission approved Kilmer's plans to convert three guesthouses on the actor's ranch property in Rowe into a lodging business.
At a meeting last month, the commission delayed a decision on Kilmer's request for the lodging business until he appeared personally to answer to criticism of his magazine quotes.
A man accused of killing another in a road-rage shooting faces another charge, which could mean more years in prison if he's convicted.
Richard Baca, 21, was facing a charge of voluntary manslaughter in the November killing of Benito Lemos, 22, in central Las Vegas. That charge carries a maximum sentence of six years in prison.
Now, he faces a charge of shooting from a motor vehicle, which has a maximum of 15 years.
The Mora County Sheriff's Department has two new certified officers in its ranks.
Americk Padilla and Amos Espinoza graduated from a law enforcement academy in Farmington last week. That brings the total number of certified officers to four, including Sheriff Roy Cordova and Sgt. Michael Benjamin.
Another deputy is expected to graduate from an academy in late August.
The department is also planning to hire another deputy.
Benjamin said Monday that having five certified deputies is likely the most the department has had at one time.
For many years, both Gary Leger and his FFA students have been recognized locally, statewide and nationally for their achievements.
But the real testament to his teaching skills came from the students themselves. He just called them "his kids."
Leger died in a car accident Wednesday night in Oklahoma; he had been attending an agricultural teachers conference.
Last Thursday morning, I received a call from a friend and associate, "Lupita, are you sitting down?"
"What's the matter? Tell me."
"Well, I don't know how to say this, but ....".
I learned that a dear friend and former colleague at Robertson High School, Gary Leger, had been in an auto accident near Oklahoma. He was airlifted to the nearest hospital, in Amarillo, but died a bit later.
The fire near Gallinas Canyon is more than 70 percent contained, officials said Monday.
The blaze, which started last Friday night, has burned 777 acres. No evacuations have been ordered.
The first is about 2.5 miles south of Gallinas Canyon, northwest of Las Vegas.
Officials said that for a time, it was the nation's top priority forest fire because of its nearness to the Gallinas watershed, which supplies 90 percent of Las Vegas' water.
San Miguel County officials aren't taking any chances during actor Val Kilmer's appearance before the County Commission today.
They are arranging for city and state police, sheriff's deputies and jailers to be on hand to provide security. The meeting is at 1:30 p.m. on the second floor of the old county courthouse.
The "Batman" actor has requested the commission approve a lodging business at his Rowe ranch.
Prosecutors have dismissed a DWI charge against a West Las Vegas school district employee who crashed a district vehicle while allegedly drunk.
In late April, the district attorney's office in Moriarty dropped the DWI charge against John J. Roybal, 41, West's head mechanic. But in a plea agreement, Roybal pleaded guilty to careless driving and weaving between lanes.
As part of the deal, Roybal must take a defensive driving course and participate in a DWI simulation program.
The Tecolote Fire south of Gallinas Canyon on the Pecos district has burned 777 but now is 40 percent contained.
No evacuations have been ordered for residents in Gallinas Canyon and El Porvenir. No buildings have been affected.
The fire is about 2.5 miles south of Gallinas Canyon; it is not near Tecolote.
More than 500 firefighters from several Western state have helped with putting out the fire. The blaze is happening in the Santa Fe National Forest.
The electric utility serving much of rural Mora and San Miguel counties is seeing a tug-of-war between members from Mora and Pecos.
During this Saturday's annual meeting, members of the Mora-San Miguel Electric Cooperative will vote on a number of proposals that could affect the amount of influence that Mora and Pecos have over the cooperative.
Eighteen proposed bylaw changes are on the ballot for the more than 10,000 members of the cooperative. Voting sites will be in Mora, Pecos and Las Vegas.
Copyright © 2015
The Las Vegas Optic is your source for local news, sports, events, and information in San Miguel County and Las Vegas, NM, and the surrounding area.


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Looking for something to do with your pet, or fun events to help support animal rescue groups? Below is a list of pet events in the WXII 12 viewing area.
If your organization is having an event that you would like posted, please SEND US AN EMAIL
Adoption and Pet Supply Drive for local shelter: 3/28/15
The Liberty Tax Service office at 405 E Dixie Drive Asheboro will have pets for adoption and serve as is a collection site for pet food and supplies for the Randolph County Animal Shelter on Saturday, March 28 from 11:30 – 1:30. Statue of Liberty costumed characters will be on hand as Liberty Tax hosts a family roadside party with refreshments and wonderful pets who need a good home. Everyone stopping by will receive a free lunch and as always, free tax advice.
The Randolph County Animal Shelter wish list of needed pet supplies includes:
- Dry Dog/Cat Food
- Bowls and Buckets
- Slip leashes
- Large/Medium Plastic Dog Crates (for cat/dog transport)
- Paper Towels
"We want to help the animals and support the work of Randolph County Animal Shelter," said Rebekka Maness, Marketing Manager of Liberty Tax Service in Asheboro, NC.
A NIGHT AT THE SALOON to benefit the SPCA of the Triad
When: Saturday, March 28th
Time: 7:00pm-11:00pm
Location: Hodgin Valley Farm
561 Hodgin Valley Road
Greensboro, NC 27313
WANTED: Joe Player for a night at the saloon! Come out and play some friendly games of luck for a great cause. We will have lots of goodies for you to enjoy while you play including a free taco bar, chocolate fountain, chili bar, other desserts and a cash bar! Raffles and prizes too! All proceeds directly benefit the SPCA of the Triad.
Tickets: $30 for Singles, $50 for Couples
For more information, CLICK HERE
AARF Doggie Easter Egg Hunt will be held on April 11, 2015.
5th Annual Bark and Wine: May 16, 2015
Bark and Wine is one of the largest rescue fundraiser's in Greensboro. This event brings together rescues, bands, and vendors from across North Carolina, for a fun-filled day of music, shopping and mingling to benefit homeless animals in the state.
When: Saturday, May 16, 2015
Time: 11:00am – 6:00pm
Cost: $5.00
Location: The Railyard (adjacent to The Worx)
For more information, to buy tickets, sponsor, be a vendor, or to volunteer, CLICK HERE
Putts for Pups – May 16, 2015
(must register by April 16, 2015)
Putts for Pups Benefit Golf Event for the Animal Protection Society of Caswell County
When: Saturday, May 16, 2015 (Rain date May 17, 2015)
Where: Caswell Pines Golf Course, Yanceyville, NC
Time: Lunch starts at 11:30, Tee off starts at 1:00
How to register your team: CLICK HERE, print and mail registration with payment to APS Caswell Shelter.
( team of 4- $50 per person by April 16th, $55 per person after April 16th)
BE A SPONSOR: Call 336-694-4921 or email us at firstname.lastname@example.com
$50 Name and Group sign at check -in
$100 name on sign at hole ( w/logo if you provide) and on Group sign
$250 name on sign at hole ( w/logo if you provide)name on beverage carts, and on Group sign
$500 – choose a team registration OR a large sign at check – in just for you; name on signs at 2 holes ( w/logo if you provide) and on Group Sign
$1000 custom sponsorship we can arrange to meet your needs
Questions – 336-694-4921 or email at email@example.com
2015 Triad Dog Games: May 16 & 17
Get your puppy dogs ready for the 2015 Triad Dog Games to be held at Tanglewood Park on May 16th and 17th.
This is a fundraising event benefiting The Sergei Foundation to help save companion pets' lives by providing veterinary financial assistance to families unable to afford emergency, life-saving care.
To learn more about the games and about Sergei Foundation, CLICK HERE
Paws in the Grove: June 27, 2015
Join Ruff Love Rescue on June 27th for the 3rd annual Paws in the Grove, at Grove Winery and Vinyards. Music, fun, food & wine. You are encouraged to bring your dogs to enjoy the evening with us.
For more information, CLICK HERE
Pets u local Object


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1996 Chevrolet Corvette Collector Edition LT4 6-Spd Coupe, Sebring Silver with black leather interior. Excellent condition. Just under 30K miles. Always garaged. Has Preferred Equipment... More
1971 Camaro RS 350 2 BBL (20,000 miles on stock rebuild), Turbo 350, This is an original Air Optioned car. (the dash has the OEM air conditioning... More
Here we have a custom 1964 Chevrolet Impala SS "low rider" style that has been mildly customized. This customized car is powered by a... More
This 1974 Corvette Convertible is an excellent weekend driver for the summer. White with tan interior and matching numbers. Automatic transmission. An overall original example... More
have a 2 owner 68 impala convertible 307 auto factory am/fm radio with 31k original,needspaint ,paint is cracked, needs roof and front seat covered,solid... More
'51Studebaker Bullet Nose front end on '78 El Camino. Front end is fiberglass. Best of both worlds, 50's style with modern chassis and power.... More
SEE MORE PHOTOS OF THIS VETTE AND 100+ OTHERS FOR SALE AT WWW.BUYAVETTE.NET! Ruby Red exterior Ruby Red leather interior Ruby Red convertible top.... More
Pay attention as you do not see these in this condition very often. This honest example of the 1964 Chevrolet Chevelle El Camino comes... More
Before we had compact pickups, we had full-sized rigs like this 1970 Chevrolet C10 step-side. The closest thing to a sports truck at the... More
This 1998 Chevrolet Camaro Z/28 has to represent the most bang for the buck in any of our showrooms right now. One, it's super... More
~CALL US FOR A DETAILED DESCRIPTION OF THE CAR! WE DO A 150+ POINT CHECK ON EACH VEHICLE AND ARE HAPPY TO SHARE... More
2D Coupe, 5.7L V8 SFI, 4-Speed Automatic with Overdrive, RWD, Black, and Black w/Leather Seat Trim. The car you've always wanted! Here it is!... More
Just in! Call or visit our website for more information.... More
Coupe, White w/Orange Stripes, Blk Int, 350 V8, Auto, Vin:10524... More
1951 Chevrolet Deluxe 2 door, This is an original car 216 c.i.3 speed standard trans, 45000 original miles, skirts.working a/m radio, and heater, no... More


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Extreme Clean Pool Products
RatherbgardeningJune 27, 2009
Does anyone use this and if so, does it do as claimed? Thanks!
Extreme Clean was pretty much a decent product (despite the high price) until one thing. Once the influx of phoaphate removers hit the market, it was pretty much understood that Extreme Clean was aluminum phosphate just like any other phosphate remover.
So, why buy the expensive product? They also stopped selling through distribution. It is a good product for water clarity and you actually use very little so that may offest the price.
As a wholesale distributor, I can tell you I have tons of the crap to unload but it is very tough due to the pricing.


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Fostering honesty in relationshipsComment
People always say that "honesty is the best policy," but following through on the old adage is where the trouble starts!
National Honesty DayComment
National Honesty Day was created so that the month of April would end with a day to focus on honesty - since it begins with a day of pranks and deceit!
Honest Tea experiment shows D.C. an honest cityComment
D.C. ranks as the nation's tenth most honest metro area—according to a recent "honesty" experiment.
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ABC7 News at 4 Now Weekdays!ABC7 is giving you MORE news in your afternoons with an extra hour, an hour earlier! Join ABC7's Leon Harris and Alison Starling for what's trending in your community and world. Every weekday afternoon at 4pm right after "The Queen Latifah Show" at 3pm on ABC7.


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Top Stories
- TRRT April 1-7 | Online Edition
- Guest Commentary: the Rockford Apartment Association
- State Roundup: NIU employee improperly reimbursed $30K
- State Roundup: Governor signs budget fix bills
- Rauner, Democratic leaders shake hands and make law
- State roundup: National guardsman and cousin arrested in terror plot
- Lawmaker says license plate readers a privacy threat
- Bryant not the first to feel impact of free agency rules
- State Roundup: Parents' group calls for standardized test opt-out bill
- Hononegah Mack: 'The best woman in the county'
Local photographer featured in Chamber publication
January 11, 2012
Brian Thomas Photography provided photography for the Rockford Chamber of Commerce's "20 People You Should Know" awards and event celebration. The portraits were used in the Chamber's One publication, Vol. 1, issue 1.
Photography services were also provided to IPSEN in Pecatonica; the images will be used to promote new and current product lines.
Nicholas Conservatory and Gardens recently hired Brian Thomas Photography to photograph its preview Gala. The company has also donated more than 20 aerial photography flights documenting the construction progression of the conservatory.
From the Jan. 11-17, 2012, issue


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San Ra•fael
Pronunciation: (san" ru-fel'; for 1 also Sp. sän rä"fä-el'), Strict Standards: Non-static method FenSites::linkTo() should not be called statically in /site/html/dictionary/san-rafael.html on line 75 [key]
1. a city in W Argentina. 70,477.
2. a city in W California, N of San Francisco. 44,700.
Random House Unabridged Dictionary, Copyright © 1997, by Random House, Inc., on Infoplease.
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text: Is Madonna a Good Choice for the Halftime Show? [POLL] By The Rob December 5, 2011 7:07 AM Share on Facebook Share on Twitter The Rob and Kat actually agree on something! Do you also think Madonna is a good choice for the Halftime Show? Madonna Facebook Sorry, you need to have javascript running to see this poll. Filed Under: kat mykals, Madonna, super bowl, the rob Category: The Rob's Radio Show Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! Best of 106.1 KISS FM Recommended For You Around the Web