Successful treatment of a generalized human papillomavirus infection with granulocyte-macrophage colony-stimulating factor and interferon gamma immunotherapy in a patient with a primary immunodeficiency and cyclic neutropenia.

BACKGROUND: We describe a patient with generalized verrucosis secondary to human papillomavirus (HPV) type 2 infection and a primary immunodeficiency and cyclic neutropenia. Treatment, which was well tolerated, included granulocyte-macrophage colony-stimulating factor and interferon gamma (IFN-gamma). In vitro assays to assess responses of T lymphocytes to mitogens (ie, proliferation assay and IFN-gamma enzyme-linked immunosorbent assay) were performed. In situ hybridization and polymerase chain reaction were used to detect HPV DNA in skin biopsy specimens. OBSERVATIONS: The T lymphocytes of the patient showed a significant (P < .05, unpaired Student t test) defect in IFN-gamma production (the basis for initiating IFN-gamma therapy). The response to immunotherapy was confirmed by using molecular methods. Six months after the completion of immunotherapy, HPV DNA was undetectable in skin samples from clinically regressed warts (according to the results of in situ hybridization and polymerase chain reaction). CONCLUSIONS: Our patient had treatment-resistant generalized verrucosis for 13 years. Treatment with granulocyte-macrophage colony-stimulating factor and IFN-gamma may have reconstituted an immune response against HPV, resulting in the dramatic regression of her widespread warts.

Heparin-binding domain of fibrin mediates its binding to endothelial cells.

Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new beta 15-42 N-terminus. To further understand the interactions between ECs and fibrin beta 15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, beta 1-21; J88B, gamma 63-78) and anti-fibrin (T2G1, beta 15-21) antibodies in an indirect enzyme-linked immunosorbent assay. To accomplish this, large, soluble fragments of fibrin were prepared by cyanogen bromide (CNBr) cleavage (fibrin-CNBr); CNBr-cleaved FBG (FBG-CNBr) served as the control ligand. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspension in a dose-dependent and saturable manner. By contrast, FBG-CNBr bound only 50% as well to EC monolayers, with no significant binding of intact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fragment E, which lacks beta 1-53. ECs bound the peptide beta 15-42-bovine serum albumin (BSA) conjugate but neither a scrambled beta 15-42 peptide conjugate nor conjugates of beta 24-42, beta 18-27, or beta 18-31. Binding of fibrin-CNBr was inhibited 54% by the beta 15-42-BSA conjugate and 17% by the B beta 1-42-BSA conjugate but not by free beta 15-42 peptide or RGDS-cell binding peptide. Binding of fibrin-CNBr was inhibited > 95% by heparin in a concentration-dependent manner; the same concentrations of heparin inhibited binding of beta 15-42-BSA by > 75% but not the dose-dependent binding of fibronection to ECs. These data suggest that in their native conformation, FBG B beta 15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of beta 15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.

Cell proliferation on fibrin: modulation by fibrinopeptide cleavage.

Fibrin forms the cohesive network of hemostatic plugs and thrombi, and it also provides the temporary matrix for initial support of healing and revascularization. Because cell proliferation is needed for revascularization after vessel injury, we have characterized structural requirements of fibrin needed to support cell proliferation on fibrin in vitro. Proliferation of cultured human endothelial cells and fibroblasts was measured by 3H-thymidine incorporation on fibrin surfaces varying in structure. Fibrin prepared with thrombin and lacking both fibrinopeptides A and B (desAB fibrin) supported proliferation of both endothelial cells and fibroblasts. In contrast, fibrin prepared with reptilase, which cleaves only fibrinopeptide A, supported significantly less proliferation. Also, fibrin prepared by thrombin treatment of fibrinogen lacking residues beta 1-42 supported only a low level of proliferation. Therefore, fibrinopeptide B cleavage and exposure of beta 15-42 enhanced proliferation of cells on fibrin. Specific proteolytic inhibitors were used to eliminate the potential mitogenic effects of residual fibrin-bound thrombin. Additional controls showed that neither catalytically inactive thrombin nor addition of the thrombin receptor-activating peptide (SFLLRNPNDKYEPF [SFLL]) stimulated proliferation on desA fibrin. The results indicate that cell proliferation on fibrin is enhanced by fibrinopeptide B cleavage and exposure of the amino terminus of the fibrin beta chain. They also show that specific structural features of the temporary fibrin matrix formed at sites of injury may modulate the proliferative response of vascular cells.

Cloning of the complete coding sequence of rat fibrinogen B beta chain cDNA: interspecies conservation of fibrin beta 15-42 primary structure.

After thrombin cleavage, the newly exposed NH2-termini of the beta chains play a role in both fibrin polymerization and fibrin interactions with cells in the process of wound healing. These physiological responses have been shown to be mediated, at least in part, by beta 15-42. To compare the sequence of the beta chain of fibrin across species, the complete coding sequence of the rat fibrinogen B beta chain cDNA was cloned (designated pRB beta 3) and characterized. The sequence newly determined from pRB beta 3 encompassed nucleotides 121-589, encoding residues 9-165 of the mature polypeptide. Significant homology of pRB beta 3 cDNA and deduced amino acid sequences was found when compared with other species' B beta chains. The rat B beta-Arg14-Gly15 thrombin cleavage site is conserved; however, the fibrinopeptide B sequences are only 50% similar when rat is compared with human. In contrast, the beta 15-42 region is 100% similar when allowing for conservative amino acid substitutions. Monoclonal antibodies (MAbs) specific for human fibrinogen B beta 1-21 (1-8C6) and human fibrin beta 15-21 (59D8 and T2G1) failed to cross-react with rat fibrinogen or fibrin by ELISA, respectively, even though thrombin conversion of rat fibrinogen to fibrin was confirmed. MAb 1-8C6 reacted with reduced and denatured human fibrinogen B beta chain by Western blotting, whereas, MAb T2G1 did not blot with reduced and denatured human fibrin beta chain. A comparative analysis of the binding affinity of the human B beta fibrin(ogen) specific MAbs with B beta fibrin(ogen) from several species suggested that amino acid residues preceding and including Arg14-Gly15 are important in the epitope of the B beta 1-21 specific MAb 1-8C6, and that residues Gly15, Leu19 and/or Lys21 play an important role in the epitope shared by the beta 15-21-specific MAbs T2G1 and 59D8.

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading.

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.

Spreading of platelets on fibrin is mediated by the amino terminus of the beta chain including peptide beta 15-42.

We have investigated the adhesion and spreading of platelets on polymerized fibrin of varying structure to identify sites that mediate these interactions. Fibrin was prepared with thrombin to remove both fibrinopeptide A (FPA) and fibrinopeptide B (FPB) and with reptilase or Agkistrodon contortrix thrombin-like enzyme (ACTE) to selectively remove FPA or FPB, respectively. Residual fibrin-bound enzymes were inhibited with D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK). Platelet adhesion was independent of fibrinopeptide cleavage and was equal on fibrin prepared with each of the three enzymes. In contrast, FPB cleavage increased spreading as quantitated by fluorescence microscopy of platelets stained for glycoprotein IIb-IIIa. The 24% +/- 4% spreading on reptilase-fibrin was significantly less than the 70% +/- 8% on thrombin-fibrin or 65% +/- 9% on ACTE-fibrin (P < .0005 for both). Protease III from Crotalus atrox venom was used to specifically cleave residues B beta 1-42 from fibrinogen to further investigate the role of the beta chain N-terminus in promoting platelet spreading. After clotting with thrombin, this fibrin derivative lacked beta 15-42 and supported significantly less spreading. A monoclonal antibody (MoAb) reactive with beta 15-21 inhibited spreading on thrombin-fibrin as did peptide beta 15-42, while control MoAbs and peptides had no significant effect. These results indicate that adhesion and spreading of platelets on fibrin are mediated by different interactions, and that spreading can be mediated by FPB cleavage and the amino terminus of the beta chain including residues beta 15-42.

Endothelial cell responses to fibrin mediated by FPB cleavage and the amino terminus of the beta chain.

Endothelial cells (EC) interact with fibrin at sites of vascular injury, thrombosis, inflammation and tumor growth, whereas they are quiescent when exposed to circulating fibrinogen. To determine the structural basis for specific interaction with fibrin we have characterized the response of EC to fibrin of varying structure. Fibrin was prepared with thrombin, which cleaves both fibrinopeptide A (FPA) and fibrinopeptide B (FPB), with Reptilase, which cleaves only FPA, and with contortrix procoagulant to cleave only FPB. Fibrin with FPB cleavage stimulated release of von Willebrand factor from EC Weibel-Palade bodies and also supported cell spreading. Involvement of the amino terminus of the fibrin beta chain in the response was shown by stimulation of von Willebrand factor release by the peptide beta 15-42. Also, fibrin prepared from a fibrinogen derivative lacking residues 15-42 of the beta chain failed to support EC spreading. EC adhesion was unaffected by the pattern of fibrinopeptide cleavage or by the removal of peptide beta 15-42 from fibrin. The results indicate that separate sites on the fibrin molecule mediate adhesion and spreading, and that the latter requires cleavage of FPB and the new amino terminus of the beta chain. They further suggest that cellular responses to fibrin are regulated by the proteolytic cleavages and conformational changes that convert fibrinogen to fibrin and may also be modulated by plasmic or elastase degradation.

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Adhesion and spreading of cultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin beta chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B beta chain. After clotting with thrombin, this fibrin derivative lacking B beta 1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 15-42 of the fibrin beta chain.

Physicians and the teaching of breast self-examination: implications from a survey at a university teaching hospital.

We surveyed 80 physicians from four specialties (Family Medicine, General Medicine, General Surgery, Ob-Gyn) to investigate how they taught breast self-examination (BSE). Only half reported personally teaching BSE. Few MDs reported routinely using techniques to assess BSE competency. Most (72 per cent) claimed no formal training in teaching BSE; 10 per cent claimed no training at all. Techniques used to teach BSE may vary, and physicians may lack the training to teach BSE.

Physicians' abilities to detect lumps in silicone breast models.

Little is known about how well physicians detect breast lumps in clinical breast examinations. We studied 80 general medicine, family medicine, general surgery, and obstetrics/gynecology physicians to determine their abilities to detect lumps in manufactured breast models. The mean number of lumps detected was 8.0 (44%), with a range of three (17%) to 15 (83%). Detection varied significantly by size (87% of 1.0-cm and 14% of 0.3-cm lumps) and hardness (56% of hard and 40% of soft lumps), but not depth; by specialty (from 50% for general internists to 40% for obstetricians), but not by level of training or experience; and by search duration (r = .59). On multiple regression analysis, only search duration was consistently associated with increased detection. Modest detection rates and wide variation suggest breast lump detection can be improved among physicians. Adequate search duration may be important for high detection rates.

Computed tomography in recurrent or metastatic colon cancer: relation to rising serum carcinoembryonic antigen.

Twenty patients with Stage Duke B or C adenocarcinoma of the colon or rectum who have undergone radical surgical resection and demonstrated rising serum carcinoembryonic antigen (CEA) during follow-up are the subject of this study. In all cases, while there was a continuous and progressive elevation of serum CEA, CT examination of the abdomen and pelvis was performed. Abnormal CT findings were demonstrated in 19 patients and included pelvic mass, liver metastases, and periaortic or mesenteric lymphadenopathy. There was one normal CT scan in a patient who subsequently developed metastases in the sacrum. Based on the observations in these patients, it is concluded that in routine follow-up after colorectal surgery, rising serum CEA should be considered a warning sign and warrants additional investigation by CT.