Fasciotomy for chronic exertional compartment syndrome of the leg: clinical outcome in a large retrospective cohort.

BACKGROUND: Chronic exertional compartment syndrome (CECS) is an overuse disorder typically affecting an athletic population. CECS is a diagnosis based on history and intracompartmental pressure (ICP) testing. CECS patients can be treated surgically by fasciotomy; however, research on the relationship between ICP and patient symptoms and also between ICP and patient-reported outcome post-fasciotomy is limited. This study aims to (1) assess functional outcome and patient satisfaction post-fasciotomy and (2) identify any potential correlation between ICP and reported levels of pain. METHODS: 138 CECS patients who had ICP measurements and subsequently underwent fasciotomy were identified from our regional service for exercise-induced lower limb extremity pain between January 2000 and March 2017. Clinical outcomes were recorded at the time of ICP testing and in the post-operative follow-up clinic. Pain was reported using a verbal rating scale (VRS) ('low', 'moderate' or 'high') or as a visual analogue score (VAS) 0-10 (0 = least painful, 10 = most painful). Spearman's ranked correlation test was used to calculate correlation between ICP and reported pain. RESULTS: A total of 138 patients were eligible for inclusion in this study (mean age 29.7 ± 9.7 years, 110 M, 28 F) of which 109 patients (VRS n = 61, VAS n = 48) reported pain level at pre- and post-operative stages. Mean pre-operative VAS score was 8.52 ± 0.71, and decreased to 0.77 ± 0.69 post-operatively. An insignificant positive correlation (r = 0.046, two-tailed p = 0.76) was found between VAS pain and ICP. A significant moderate positive correlation (r = 0.497, two-tailed p = 0.01) was found between VRS pain and ICP. CONCLUSION: Fasciotomy significantly reduces pain and increases activity levels in CECS patients. ICP was found to positively correlate with patient-reported pain.

Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.

Synthetic peptides from the circumsporozoite proteins of Plasmodium falciparum and Plasmodium knowlesi recognize the human hepatoma cell line HepG2-A16 in vitro.

Several lines of evidence have emphasized the importance of the malaria circumsporozoite (CS) protein as a factor in sporozoite invasion of the hepatocyte; however, the specific mechanism of cell recognition and invasion has not been explained. In this study we present evidence that a highly conserved region of the CS protein immediately adjacent to the repeat region, the N1 region, specifically recognizes receptors on the human hepatoma cell line HepG2-A16 under conditions where invasion by sporozoites can occur. Peptides consisting of sequences from the repeat region or of the more extensive N2 region showed no such specific association. Antibody against the N1 peptide could inhibit sporozoite invasion in vitro. Covalent coupling of radiolabeled N1 peptide to HepG2-A16 cells identified two hepatic cell proteins to be closely associated with the peptide. We suggest that these proteins could act as receptors or mediators, via the N1 region of the CS protein, for the P. falciparum sporozoite in the process of invasion of the hepatocyte.

Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria.

We show here an effective and novel approach to engineer peptide-based vaccines using a chemically defined system, known as multiple peptide antigen systems (MAPs), to protect an inbred mouse strain from infection against rodent malaria. 10 mono- and di-epitope MAP models containing different arrangements and stoichiometry of functional B and/or T helper cell epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize A/J mice. While these mice did not respond to the mono-epitope MAP bearing only the B or T epitope, very high titers of antibody and protective immunity against sporozoite challenge were elicited by di-epitope MAPs, particularly those with the B and T epitopes in tandem and present in equimolar amounts. These results, obtained in a well-defined rodent malaria model, indicate that MAPs may overcome some of the difficulties in the development of synthetic vaccines, not only for malaria but also for other infectious diseases.

Detection of transforming growth factor alpha in normal, malignant, and hyperproliferative human keratinocytes.

Transforming growth factor alpha (TGF-alpha) is a 50-amino acid peptide, previously demonstrated only in transformed cell lines and human tumors, which is structurally homologous to epidermal growth factor (EGF). TGF-alpha expression in keratinocytes from normal individuals, patients with psoriasis, and patients with malignant skin diseases was investigated using an mAb raised against synthetic human TGF-alpha. mAb A1.5 reacted with TGF-alpha, but not EGF, in a sensitive ELISA. Keratinocytes in eight nodular basal cell carcinomas, one morpheic basal cell carcinoma, and one squamous cell carcinoma demonstrated intense membranous immunoperoxidase staining with mAb A1.5. Of even greater interest was the observation that the overlying normal epidermis, as well as the epidermis from five normal skin specimens, were stained by the mAb. Keratinocytes in plaques from 18 psoriasis patients were more intensely stained than those from normal skin. Cultured normal keratinocytes demonstrated membranous staining with mAb A1.5. Absorption of mAb A1.5 with synthetic human TGF-alpha completely removed the reactivity of mAb A1.5 with both basal cell tumors and normal epidermis. The demonstration of TGF-alpha in normal keratinocytes suggests that it plays a role in normal keratinocyte growth, wound healing, and in the pathogenesis of acanthosis.

Synthetic peptide vaccine confers protection against murine malaria.

A synthetic peptide, (DPPPPNPN)2D, representing a subunit of the repeat domain of the Plasmodium berghei circumsporozoite protein, was conjugated to tetanus toxoid using bisdiazobenzidine. Immunization of mice and rats with the conjugate induced high serum titers of antibodies to the parasite, and most of the animals were completely protected from malaria infection when challenged with sporozoites.

Physiological effects of transforming growth factor in the newborn mouse.

Transforming growth factor-type alpha accelerated incisor eruption and eyelid opening in the newborn mouse and also retarded the growth rates of hair and body weight when administered in high dosage (0.7 to 4 micrograms per gram of body weight). The results of whole animal studies indicate that transforming growth factor-type alpha and epidermal growth factor do not differ significantly in these effects and suggest that transforming growth factor-type alpha may be important in immature animals.

Circumsporozoite protein of Plasmodium vivax: gene cloning and characterization of the immunodominant epitope.

The gene encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium vivax has been cloned. The deduced sequence of the protein consists of 373 amino acids with a central region of 19 tandem repeats of the nonapeptide Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly. A synthetic 18-amino acid peptide containing two tandem repeats binds to a monoclonal antibody directed to the CS protein of Plasmodium vivax and inhibits the interaction of this antibody with the native protein in sporozoite extracts. The portions of the CS gene that do not contain repeats are closely related to the corresponding regions of the CS genes of two simian malarias, Plasmodium cynomolgi and Plasmodium knowlesi. In contrast, the homology between the CS genes of Plasmodium vivax and Plasmodium falciparum, another malaria parasite of humans, is very limited.

Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria.

Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.

Long-term high-titer neutralizing activity induced by octameric synthetic HIV-1 antigen.

A titer for homologous viral neutralization activity (greater than 1:19,683) was observed after a 3.5-year immunization period with an octameric, branching peptide representing the principal neutralizing determinant (PND) of the human immunodeficiency virus-1IIIB envelope protein. Booster immunizations elicited persistent and potent antibodies in guinea pigs, exceeding responses produced by a conventional bovine serum albumin conjugate by 100-fold. Peptide length, central presentation of a conserved sequence, and inclusion of an upstream sequence contributed to immunogenicity. Titers (greater than 1:1,000) of heterotypic neutralizing antibodies also developed. Octameric PND peptides are a promising approach for an acquired immunodeficiency syndrome (AIDS) vaccine.

Biochemical and functional characterization of Epstein-Barr virus-encoded BARF1 protein: interaction with human hTid1 protein facilitates its maturation and secretion.

EBV BARF1 gene encodes a secretory protein with transforming and mitogenic activities. In this report, the post-translational modification, folding, maturation and secretion of BARF1 are systematically studied by site-directed mutagenesis and overexpression of the protein in mammalian cells using the vaccinia/T7 system. The protein was shown to be post-translationally modified by N-linked glycosylation on the asparagine 95 residue. This modification was confirmed to be essential for the maturation and secretion of the protein. Analysis of the four cysteine residues by site-directed mutagenesis demonstrated that cysteine 146 and 201 were essential for proper folding and secretion of the protein. To search for human proteins involved in the maturation process of the protein, a yeast two-hybrid screening was carried out using the BARF1 sequence from amino acids 21-221 (BARF1Delta) as bait, leading to the identification of human hTid1 protein as a potential interacting protein. This interaction was subsequently confirmed by coimmunoprecipitation and dual immunofluorescent labeling of cells coexpressing BARF1 and hTid1, and the interaction domain in hTid1 was mapped to amino acids 149-320. Interestingly, coexpression of BARF1 with hTid1 demonstrated that hTid1 could promote secretion of BARF1, suggesting that hTid1 may act as a chaperone to facilitate the folding, processing and maturation of BARF1.

Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family.

Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.

The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes.

Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells.

Coronavirus envelope (E) protein is a small integral membrane protein with multi-functions in virion assembly, morphogenesis and virus-host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS-CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N- and C-termini of the SARS-CoV E protein are exposed to the cytoplasmic side of the membranes (N(cyto)C(cyto)). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N-terminus exposed luminally and the C-terminus exposed cytoplasmically (N(exo(lum)-)C(cyto)). Intriguingly, a minor proportion of the SARS-CoV E protein was found to be modified by N-linked glycosylation on Asn 66 and inserted into the membranes once with the C-terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS-CoV E protein may provide a useful clue to the pathogenesis of SARS-CoV.

Elevation of transforming growth factor alpha and its relationship to the epidermal growth factor and alpha-fetoprotein levels in patients with hepatocellular carcinoma.

A radioimmunoassay for transforming growth factor alpha (TGF-alpha) using synthetic rat sarcoma transforming growth factor and its rabbit polyclonal antibody has been developed. Using radioimmunoassays, the urinary TGF-alpha and epidermal growth factor (EGF) concentrations in 31 patients with hepatocellular carcinoma (HCC), 15 probable HCC, four metastatic liver cancer, and 33 age, sex-matched healthy controls were determined. For the first time, we have shown that the average TGF-alpha concentration for HCC patients was 21.5 +/- 20.3 micrograms per g creatinine, significantly higher than that of healthy subjects, 4.9 +/- 2.8 micrograms per g creatinine (P less than 0.001). There was no statistical difference in the level of EGF between HCC patients and controls (40.9 +/- 29.3 versus 46.2 +/- 16.6 micrograms per g creatinine; P greater than 0.05). The ratio of EGF/TGF-alpha between HCC patients (3.37 +/- 4.42) and controls (15.5 +/- 13.0) was significantly different (P less than 0.001). Among patients, 65% (20 of 31) of HCC cases and 87% (13 of 15) of probable HCC cases showed a marked elevation of TGF-alpha levels. We found only 16% (five of 31) of HCC cases with increased EGF level. EGF excretion was inversely age related. Serum total protein concentration and alkaline phosphatase activity were positively correlated to EGF concentration (r = 0.522, P less than 0.01 and rt = 0.393, P less than 0.05, respectively). There was no correlation between biochemical functions of liver and TGF-alpha concentration in HCC patients. Our results also suggested that TGF-alpha may be a useful complementary tumor marker for management of patients with clinical manifestation of HCC who have low alpha-fetoprotein levels.

Dissecting intracellular signaling pathways with membrane-permeable peptides.

Peptides can be designed that mimic protein interaction motifs and thus, can be used to specifically and selectively block particular steps in signal transduction cascades where protein interactions have been previously identified. This protocol describes methods to synthesize peptides coupled to a membrane-permeable sequence (MPS), designed from the signal sequence of Kaposi fibroblast growth factor, which has been previously shown to translocate covalently attached cargo peptides across the cell membrane. To increase efficiency, yield, and versatility in the preparation of these membrane-permeable peptides, a modular synthesis strategy based on two unprotected peptide segments was designed. The modular synthesis strategy allows the MPS and functional peptides to be synthesized separately. In this manner, the functional domain of a peptide or protein, synthesized by traditional fluoroenylmethyloxy-carbonyl (Fmoc) chemistry or derived from recombinant expression, may be purchased commercially to expedite synthesis. Subsequently, the MPS domain may be attached to any functional domain using a one-step conjugation reaction. This protocol provides detailed methods for peptide synthesis, activation of the MPS, and the subsequent conjugation protocol.

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance.

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.

Lipophilic multiple antigen peptide system for peptide immunogen and synthetic vaccine.

We describe the development and structural requirements of a new lipophilic multiple antigen peptide (lipoMAP) system for immunogens that contains a built-in lipophilic adjuvant and has the ability to elicit cytotoxic T-lymphocytes (CTLs). In addition to the peptide antigens of choice at the amino terminus, the basic lipoMAP design consists of three components: a tetravalent symmetrical core matrix containing two levels of branching beta-alanyl-lysine as a building unit, a hydrophilic Ser-Ser dipeptide linker, and at the carboxyl terminus, palmitoyl lysines (PL) with alternating chirality. An 18-residue peptide from the third variable region in the gp120 of HIV-1 was used as antigen in eight models for a structure-function study. Alternating palmitoyl lysine (PL) was introduced as the lipid anchor and built-in adjuvant because D and L Lys (Pal) was found via molecular modeling to best mimic phosphatidylcholine and thus provide the most stable peptide antigens on the ordered lipid membranes. The requirements of the palmitoyl lysines and the L-Ser-L-Ser linker were crucial, since replacement with palmitoyl serines or L-Ser-D-Ser linkers led to a marked decrease in immune response. The stoichimetric ratio of PL vs MAP was also important. Multiple antigen peptide (MAP) constructs without the lipophilic PLs, those that were underlipidated and contained one PL, or those that were overlipidated containing four PLs, were ineffective. LipoMAPs containing three palmitic acids elicited significant humoral responses in oil-based emulsion and liposomes, but not in water or alum formulations. LipoMAP containing only two PLs was found best to be incorporated in liposomes and elicited a significant immune response and cytotoxic T-lymphocytes (CTLs). These models were compared favorably with a preparation using tripalmitoyl-S-glyceryl cysteine (P3C) as the lipid anchor. We also developed a modular synthesis of MAP-P3C that incorporated P3C as a premade unit containing a thiopyridine, which simplified the overall scheme and minimized oxidation during stepwise peptide synthesis. This lipoMAP model is a new addition to the design of our macromolecular assemblage approach mimicking peptide antigens on the surface of micro-organisms. It may be a potentially useful approach to the design of a synthetic vaccine for humans.

Chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system.

We described a novel and simple approach to prepare chemically unambiguous peptide immunogen using the multiple antigen peptide (MAP) approach. This approach requires the conjugation of two purified components: a chloroacetylated oligomeric lysine core matrix and a synthetic peptide containing cysteine at either the carboxyl or amino terminus. The resulting MAP is structurally unambiguous and contains a quantifiable amount of peptide antigens. Furthermore, this method also provides a flexible strategy to link a peptide antigen to the core matrix at the desirable orientation to mimic the native molecule. The carboxyl fragment 43-50 of human transforming growth factor alpha (TGF alpha) was used as a test model for this approach. Antipeptide antibodies did not recognize the "reverse immunogen" in which the peptide was attached to the MAP core matrix at a reverse orientation. To determine the specificity of the antibodies, we used two series of point-substituted TGF alpha analogs containing either alanine or the corresponding D-amino acid replacement to map the antigenic site. The alanine analogs were used to determine the contribution of the side chain while the D-amino acid analogs were used to determine the importance of backbone conformation. The antigen site was found to consist of four residues (Asp47-Leu48-Leu49-Ala50) at the distal end of the peptide-MAP conjugate. The results provide a clear explanation for the specificity of the antipeptide antibodies and their failure to recognize the "reverse immunogen" since the distal and the flexible end of the peptide-MAP construct constitutes the antigenic site. Furthermore, our results also suggests a strategy of placing the antigenic portion of a short-peptide at the distal end in the MAP approach to prepare immunogen.

A biomimetic strategy in the synthesis and fragmentation of cyclic protein.

This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine-knot motif with three disulfide bonds and an end-to-end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiol-thiolactone exchanges to arrive at an alpha-amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S,N-acyl migration to form the cyclic protein. A two-step disulfide formation strategy is employed for obtaining the desired disulfide-paired products. Partial acid hydrolysis through intramolecular acyl transfer of X-Ser, X-Thr, Asp-X, and Glu-X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine-knot motif of 1-4, 2-5, and 3-6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and characterization of large end-to-end cyclic peptides and small proteins.