Molecular requirements for T cell recognition by a major histocompatibility complex class II-restricted T cell receptor: the involvement of the fourth hypervariable loop of the Valpha domain.

The role of two central residues (K68, E69) of the fourth hypervariable loop of the Valpha domain (HV4alpha) in antigen recognition by an MHC class II-restricted T cell receptor (TCR) has been analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11, acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68) and glutamic acid 69 (E69) of HV4alpha have been mutated both individually and simultaneously to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated TCRs to Ac1-11-I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68 plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.

Administration of myostatin does not alter fat mass in adult mice.

AIM: Myostatin, a member of the TGF-beta superfamily, is produced by skeletal muscle and acts as a negative regulator of muscle mass. It has also been suggested that low-dose administration of myostatin (2 mug/day) in rodents can reduce fat mass without altering muscle mass. In the current study, we attempted to further explore the effects of myostatin on adipocytes and its potential to reduce fat mass, since myostatin administration could potentially be a useful strategy to treat obesity and its complications in humans. METHODS: Purified myostatin protein was examined for its effects on adipogenesis and lipolysis in differentiated 3T3-L1 adipocytes as well as for effects on fat mass in wild-type, myostatin null and obese mice. RESULTS: While myostatin was capable of inhibiting adipogenesis in 3T3-L1 cells, it did not alter lipolysis in fully differentiated adipocytes. Importantly, pharmacological administration of myostatin over a range of doses (2-120 mug/day) did not affect fat mass in wild-type or genetically obese (ob/ob, db/db) mice, although muscle mass was significantly reduced at the highest myostatin dose. CONCLUSIONS: Our results suggest that myostatin does not reduce adipose stores in adult animals. Contrary to prior indications, pharmacological administration of myostatin does not appear to be an effective strategy to treat obesity in vivo.

Biophysical and structural studies of TCRs and ligands: implications for T cell signaling.

The availability of soluble alphabeta TCRs and the individual chains has now made it possible to carry out structural studies of these molecules and analyze their molecular interactions with peptide-MHC ligands. Recent X-ray crystallographic structures of TCR alpha and beta chains have finally established their structural similarity with the lg molecules. Kinetic measurements of the interaction between TCRs and their ligands have provided strong evidence in favour of an affinity/avidity model for T cell activation in the periphery as well as during development in the thymus.

Linkage of genetic markers on human chromosomes 20 and 12 to NIDDM in Caucasian sib pairs with a history of diabetic nephropathy.

The potential contribution of maturity-onset diabetes of the young (MODY) genes to NIDDM susceptibility in African-American and Caucasian NIDDM-affected sibling pairs with a history of adult-onset diabetic nephropathy has been evaluated. Evidence for linkage to NIDDM was found with polymorphic loci that map to the long arms of human chromosomes 20 and 12 in regions containing the MODY1 and MODY3 genes. Nonparametric analysis of chromosome 20 inheritance data collected with the MODY1-linked marker D20S197 provides evidence for linkage to NIDDM with a P value of 0.005 in Caucasian sib pairs using affected sibpair (ASP) analyses. Non-parametric analysis of chromosome 12 inheritance data collected with the MODY3-linked markers D12S349 and D12S86 provides evidence for linkage to NIDDM with P values of 0.04 and 0.006, respectively, in Caucasian sib pairs using similar analyses. No evidence for linkage of MODY1 and MODY3 markers to NIDDM in African-American sib pairs was observed. In addition, no evidence for linkage to MODY2 (glucokinase-associated MODY) was observed with either study population. Results of multipoint maximum logarithm of odds (LOD) score analysis were consistent with the ASP results. A maximum LOD score of 1.48 was calculated for linkage to MODY1-linked loci and 1.45 to MODY3-linked loci in Caucasian sib pairs. Tabulation of allele sharing in affected sib pairs with D20S197 and D12S349 suggests that affected sibling pairs may inherit susceptibility genes simultaneously from chromosome 20 and chromosome 12. The results suggest that genes contributing to NIDDM in the general Caucasian population are located in the regions containing the MODY1 and MODY3 genes.

Diffuse alveolar hemorrhage and pulmonary capillaritis due to propylthiouracil.

Propylthiouracil (PTU) has recently been observed to be associated with antineutrophil cytoplasmic antibody (ANCA)-positive small vessel vasculitis, resulting in crescentic glomerulonephritis and, infrequently, diffuse alveolar hemorrhage (DAH). We describe a case of a 23-year-old pregnant woman who developed a perinuclear ANCA and antimyeloperoxidase-positive small vessel vasculitis manifesting as DAH and crescentic glomerulonephritis after she began taking PTU. An open lung biopsy was consistent with pulmonary capillaritis. She responded to corticosteroid therapy and discontinuation of PTU. DAH can be caused by pulmonary capillaritis, bland hemorrhage, or diffuse alveolar damage. To our knowledge, this represents the first documentation of an underlying pulmonary capillaritis in a case of PTU-induced DAH.

Monoclonal antibodies against flagellar antigen of Salmonella typhi.

Two hybrid cell clones secreting monoclonal antibodies against flagellar antigen isolated from Salmonella typhi, were produced and characterized. The antibodies bound specifically to the flagellar strain of S. typhi and did not show any reactivity with a flagellar S. typhi or with flagellar strains of S. dublin, S. paratyphi A, S. paratyphi B, S. typhimurium, E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The antibodies recognized a determinant present on a group of proteins migrating between 45 Kd and 60 Kd. These monoclonal antibodies would be useful reagents for clinical and epidemiological studies.

Hospital morbidity due to post-operative infections in obstetrics & gynecology.

OBJECTIVE: To study the incidence and risk factors for postoperative infection following cesarean sections and major gynecological surgery. METHODS: Postoperative infection was documented in the specified registers in all patients following cesarean sections and major gynecological surgery from January 1997 to December 1998. This study was a part of the prospective analysis of hospital-based morbidity and mortality in the Department of Obstetrics and Gynecology. RESULTS: There were a total of 89 cases of postoperative infections amongst 4,032 patients undergoing major operations giving an overall infection rate of 2.2%. The morbidity due to infections was 3.3% in cesarean sections and 0.9% in major gynecological surgery. Abdominal hysterectomies had a higher infection rate than vaginal surgery. The most common causative organisms isolated were Enterococcus, Staphylococcus and Klebsiella species. CONCLUSION: It was found that vaginal flora was a significant source of contamination during surgery, which could be minimised by local sterilisation methods. The high infective morbidity in abdominal hysterectomies needs further analysis of the risk factors. Infection surveillance with a regular review of antibiotic protocols is recommended.

Profile of injuries arising from the 2005 Kashmir earthquake: the first 72 h.

BACKGROUND: The Kashmir Earthquake of October 8, 2005 had widespread destructive effects with in excess of 86,000 people killed and over 80,000 severely injured. Most hospitals were destroyed and limited facilities were available for medical service in the immediate aftermath. A small military hospital in Forward Kahuta, Pakistan, remained functional and was inundated with severely injured patients over 72h. METHODS: A retrospective review of medical records to document the injury patterns, subsequent treatment, infections and logistical requirements that occurred following this earthquake. RESULTS: One thousand five hundred and two patients were triaged over 72h. Four hundred and sixty eight (31.1%) patients required admission. Three hundred and nineteen (68.2%) patients were managed non-operatively and 149 (31.8%) required a procedure under general anaesthesia. The most common type of injuries were: superficial lacerations (64.9%); fractures (22.2%); and soft tissue contusions/sprains (5.9%). There were 266 major injuries to the extremities (40.1% upper limb; 59.9% lower limb). Six patients had significant abdominal injuries, 66.6% of these required urgent laparotomy. 14.8% had clinically relevant infections at follow-up requiring surgical debridement or antibiotic therapy. CONCLUSIONS: Disaster response in the early phase of earthquake relief is complex, with local facilities often overwhelmed and damaged. Limb injuries are most likely; however facilities should have clear plans to deal with severe trauma including head injuries and penetrating abdominal trauma. Coordinated effort is required for success, with lessons learnt to improve future disaster management.

Earthquake injuries and the use of ketamine for surgical procedures: the Kashmir experience.

The October 8, 2005 earthquake in Northern Pakistan had widespread destructive effects throughout the northern subcontinent. Large numbers of people were killed or severely injured and many medical services destroyed. This report describes the experience of the only standing surgical hospital in the Kashmir region of Bagh District. More than 1,500 people were triaged in 72 hours, many critically injured; 78.4% of patients had upper or lower limb injuries; 50.3% of patients had fractures, mainly closed; 37% of patients required extensive wound debridements. A total of 149 patients received emergency surgery using ketamine anaesthesia with benzodiazepine premedication. This was found to be safe, effective and with a low incidence of major adverse effects. We recommend that ketamine anaesthesia be encouraged in disaster area surgery, particularly in under-resourced regional centres.

Identification of specific recognition molecules on murine mononuclear phagocytes and B lymphocytes for Vi capsular polysaccharide: modulation of MHC class II expression on stimulation with the polysaccharide.

Vi bacterial polysaccharide is a homopolymer of alpha 1-4 N-acetyl polygalacturonic acid with variable O-acetylation at position C-3 and forms a capsule around many bacteria. It has been referred to as the virulence factor of Salmonella typhi and is also a candidate vaccine against typhoid fever. The present study reports the interaction of this polysaccharide with murine mononuclear phagocytes and lymphocytes, and with human monocytes. Vi showed a dose-dependent binding to the murine monocyte cell lines WEHI-274.1 and J774. This binding was abrogated if the polysaccharide was deacetylated, suggesting involvement of acetyl groups in this interaction. Vi also bound to the murine B-cell lymphoma line A20, to peritoneal exudate cells and to a lesser degree to spleen cells and thymocytes from BALB/c mice. The polysaccharide also interacted with the human histiocytic lymphoma line U937 but not with the human monocyte cell line THP-1. Stimulation with Vi led to up-regulation of surface major histocompatibility complex (MHC) class II expression on A20 cells. Immunoprecipitation of Vi-bound molecules from cell surface biotinylated A20 and WEHI-274.1 revealed two bands with MW of about 32,000 and 36,000. The study demonstrates that Vi capsular polysaccharide can interact with mononuclear phagocytes and lymphocytes through specific cell surface molecules and modulate MHC class II expression.

Activation of a T cell hybridoma by an alloligand results in differential effects on IL-2 secretion and activation-induced cell death.

The molecular nature of the interaction of T cell receptors (TCR) with alloligands is not well understood. Although a role for groove-bound peptide(s) has been clearly demonstrated for major histocompatibility complex (MHC) class I alloreactivity, this has not been established for MHC class II-induced alloresponses. In the present study, we have analyzed the interaction of a nominal peptide-self MHC complex and of an alloligand with their cognate TCR (1934.4 TCR for autoantigen recognition and qCII85.33 TCR for allorecognition). Our results demonstrate that 1934.4 TCR recognition of the N-terminal epitope of myelin basic protein (Ac1-11, Ac=acetylated at position 1) complexed with the MHC class II molecule I-A(u) involves contacts with both chains of the MHC molecule. In contrast, qCII85.33 TCR recognition of an allopeptide:I-A(u) complex appears to predominantly involve the beta chain of the MHC molecule. Thus, the two TCR appear to have different footprints on the I-A(u) molecules. Unexpectedly, this differential involvement of the two chains of the I-A(u) molecule affects activation induced cell death, with allostimulation resulting in poor induction of FasL expression and relatively low levels of apoptosis. Significantly, stimulation of cognate T cells with alloantigen or autoantigen results in similar levels of IL-2 secretion. The reduced apoptosis of T cells in response to allostimulation may be one of the mechanisms that favors the expansion of a relatively large repertoire of alloreactive T cells.

Malignant fibrous histiocytoma (MFH) arising within a cholecystectomy incision: a cause-and-effect relationship is possible between previous surgery and the development of a malignant fibrous histiocytoma within the operative site.

We present a report of a patient who, after an uneventful cholecystectomy, developed an incisional mass that proved to be a malignant fibrous histiocytoma and had to be excised. Five months later, the patient developed a similar incisional mass, which was shown to be a malignant fibrous histiocytoma and again had to be excised. The rarity of such a case and the details of the patient's case history are reported. The implications of a possible relationship between previous surgery, the patient's healing response, and the development of these tumors are discussed.

Monoclonal antibodies delineate multiple epitopes on the O antigens of Salmonella typhi lipopolysaccharide.

Fifteen monoclonal antibodies (MAbs) directed against Salmonella typhi were produced and characterized. The specificities of the antibodies were determined by their binding patterns in an enzyme immunoassay, with a panel of lipopolysaccharides isolated from different bacteria. Seven MAbs reacted with S. typhi, Salmonella enteritidis, and Salmonella dublin (all belonging to serogroup D). One MAb also reacted with Salmonella paratyphi A and S. paratyphi B. Five MAbs reacted with S. typhi, S. enteritidis, S. dublin, and S. paratyphi B. Two MAbs did not bind to any lipopolysaccharide but showed reactivity with bacterial sonic extracts isolated from S. typhi, S. paratyphi A, S. paratyphi B, Escherichia coli, and Shigella sonnei. These antibodies would be helpful in studying the complexity of antigenic determinants expressed by S. typhi and the nature of the antibody response during typhoid and paratyphoid fevers and also in the diagnosis of the disease.

Monoclonal antibodies against two discrete determinants on Vi capsular polysaccharide.

Vi is a linear homopolymer of 1,4 N-acetyl galactosaminuronic acid. It is present in S. typhi and some other members of Enterobacteriaceae. Vi antigen of S. typhi has been associated with the virulence of the organism and a vaccine based upon this antigen has been found to confer immunity against typhoid. In this paper, we report production and characterization of four hybrid cell clones secreting monoclonal antibodies against Vi capsular polysaccharide. Binding analysis using different derivatives of Vi showed that three monoclonal antibodies reacted with the antigenic determinant constituted by O-acetyl group and one recognised the epitope constituted by N-acetyl and carboxyl groups together. All the antibodies bound to Vi positive strains of S. typhi and did not show any significant reactivity with Vi negative strains of S. typhi, S. paratyphi A, S. paratyphi B and E. coli. Besides their utility in studying the sub-specificity of antibodies produced after vaccination with Vi, these antibodies would be helpful in the diagnosis of typhoid fever.

Anti-ROS-DNA monoclonal antibody as molecular probe for oxidative DNA damage.

Modification of 400 bp (approximate size) calf thymus DNA with OH radical resulted in lowered Tm, modification of thymine (58.3%), guanine (20%) and single strand breaks. Monoclonal antibodies (mAb) generated against ROS-DNA were of IgG1 subclass. The mAb showed strong binding to ROS-DNA and ROS-modified bases and polymers, in particular, of thymine. The mAb, therefore, preferentially recognizes ROS-modified epitopes on nucleic acids. Distinct binding to DNA isolated from aged, but not from normal humans by the monoclonal antibody was observed. The antibody effectively recognized oxidative lesions in DNA from cancer patients. These studies demonstrate the potential application of the mAb as an immunochemical probe to detect oxidative DNA lesions.

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases.

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.

Sandwich enzyme immunoassays for detection of Salmonella typhi.

Enzyme immunoassays were developed using monoclonal antibodies raised against somatic (O), flagellar (H) and capsular (Vi) antigens of Salmonella typhi. The assay based on anti-O monoclonal antibodies could specifically detect S. typhi and soluble lipopolysaccharide (LPS) isolated from S. typhi. Anti-H MoAbs detected motile S. typhi and soluble flagellar antigen. Monoclonal antibodies against capsular polysaccharide could detect Vi-containing S. typhi as well as soluble Vi antigen. The three assays reported here detected S. typhi with 100% sensitivity in blood culture broths obtained from bacteriologically confirmed typhoid patients and were negative with blood specimens containing Salmonella senftenberg, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis or Streptococcus (alpha-hemolytic) derived from patients with pyrexia. The assays, however, did not demonstrate the presence of soluble antigens in sera and urine samples obtained from typhoid patients.