Vascularised fibula osteocutaneous flap for cervical spinal and posterior pharyngeal wall reconstruction

We report a case of vascularised fibula osteocutaneous flap used for composite cervical spinal and posterior pharyngeal wall reconstruction, in a patient with recurrent skull base chordoma, resected by an anterior approach via median labio-mandibular glossotomy approach. Bone stability and pharyngeal wall integrity were simultaneously restored     

Epidemiology and control of malaria in Malaysia.

In spite of more than 30 years of control activities, malaria continues to be the most important parasitic infection in Malaysia, accounting for 39,189 confirmed cases in 1991, giving an annual parasite incidence rate of 2.2 per 1,000 population. Some factors contributing to the continued transmission of malaria are the development of drug resistant Plasmodium falciparum, changes in vector behavior, and ecological changes due to socio-economic reasons. Malaria parasite rates are higher among the Aborigines, land scheme settlers and those in intimate contact with the jungle, like loggers. There has been no substantial change in the proportion of the three common malaria species responsible for infections, P. falciparum, P. vivax, P. malariae and mixed infections accounting for about 70%, 28%, 1% and 1%, respectively of all infections. Drug resistant P. falciparum is unevenly distributed in Malaysia, but based on clinical experience and in vitro drug sensitivity studies, chloroquine resistance is frequently encountered. There has been clinical and laboratory evidence of resistance to sulfadoxine/pyrimethamine combination as well as quinine, but all these have so far been successfully treated with a combination of quinine and tetracycline. The eradication of the disease is impossible in the near future but there is confidence that with better surveillance techniques and the use of alternative control measures like permethrin impregnated bed-nets to complement existing ones, the target of bringing down the annual parasite incidence to 2 per 1,000 population during the Sixth Malaysian Plan period (1991-1995) can be achieved.     

Development of a Forced Degradation Profile of Alosetron by Single Mode Reversed-Phase HPLC,LC-MS,and its Validation

Determination of alosetron in the presence of its degradation products was studied and validated by a novel HPLC method. The separation of the drug and its degradation products was achieved with the Jones Chromatography C₁₈ analytical column (150 mm x 4.6 mm; 3 µm) with a stationary phase in isocratic elution mode. The mobile phase used was 0.01 M ammonium acetate, pH-adjusted to 3.5 with glacial acetic acid and acetonitrile in the ratio of 75:25 (V/V) at a flow rate of 1 ml/min and UV detection was carried out at 217 nm. Further, the drug was subjected to stress studies for acidic, basic, neutral, oxidative, and thermal degradations as per ICH guidelines and the drug was found to be labile in base hydrolysis and oxidation, while stable in acid, neutral, thermal, and photolytic degradation conditions. An MS study has been performed on the major degradation products to predict the degradation pathway of alosetron. The method provided linear responses over the concentration range of 100–1500 ng/ml and regression analysis showed a correlation coefficient value (r²) of 0.994. The LOD and LOQ were found to be 1 ng/ml and 3 ng/ml, respectively. The developed LC method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.     

Apoptotic-related protein expression in the diaphragm and the effect of dopamine during inspiratory resistance loading

Dopamine (DA) is a free radical scavenger that attenuates apoptosis. We studied the effects of normal saline (NS) and DA on diaphragm apoptotic protein expression following 60 min of inspiratory resistance loading in rats. We tested for 27 apoptotic-related proteins and found 12 in the diaphragm. Of the 12 proteins, superoxide dismutase copper zinc (SOD [CuZn]) and proprioceptive event related potential (PERP) were significantly higher in the DA group than in the NS and sham groups (p = .002, p = .007). DA group diaphragms had significantly greater expression of SOD (CuZn) than the NS (p = .005) and sham group diaphragms (p = .003). Likewise, the DA group had significantly greater expression of PERP than the NS group (p = .008). These results suggest that DA decreases diaphragm apoptosis through elevated expression of SOD (CuZn). The identification of 12 apoptotic-related proteins will assist investigators as they study diaphragm apoptosis.     

One-Step 2-Minute Test To Detect Typhoid-Specific Antibodies Based on Particle Separation in Tubes

Typhoid fever is caused by Salmonella typhi. Detection of anti-S. typhi antibodies in the patient is a useful diagnostic aid. Among the various methods developed over the years for this purpose, the Widal test, based on bacterial agglutination, has remained the most widely used, even though it is neither specific nor sensitive. Its popularity stems from the fact that it is simple to use and inexpensive. We describe a new test which also uses a simple one-step procedure but is more rapid and accurate than the Widal. The new test (TUBEX) detects anti-Salmonella O9 (both immunoglobulin M [IgM] and IgG) antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S. typhi lipopolysaccharide (LPS) conjugated to magnetic latex particles. The reactants are mixed in a specially designed microtube for 2 min, and the result is read based on the resultant color of the supernatant following forced sedimentation of the magnetic beads. In the absence of inhibitory antibodies, there is a color change (from blue to red) due to cosedimentation of the indicator particles with the magnetic particles, whereas if these antibodies are present, they prevent such a change to a degree dependent on their concentration. Preliminary examination of TUBEX using the anti-O9 MAb and irrelevant MAbs as inhibitors revealed the test to be specific and reproducible, with an analytical sensitivity of 16 μg per ml of antibody. The reagents remained stable for at least 9 months when kept at 4°C. In the examination of 16 stored sera obtained from 14 patients with proven cases of typhoid fever and 78 serum samples from 75 subjects without typhoid fever, TUBEX was found to be 100% sensitive and 100% specific. The nontyphoid group comprised 26 healthy blood donors, 30 antinuclear antibody (ANA)-negative patients, 9 ANA-positive patients, of whom 1 was positive for anti-DNA antibody, 4 typhus patients, and 6 septicemic patients. In addition, the sera obtained from 11 patients clinically diagnosed as having typhoid fever were all positive in the test. The TUBEX results correlated to some extent, albeit insignificantly (r = 0.38, P = 0.07), with those of an enzyme-linked immunoassay (ELISA) which used a similar detection format (inhibition) and reagents (S. typhi LPS and anti-O9 antibody). TUBEX correlated very well with ELISAs which detected anti-S. typhi LPS IgM (r = 0.58, P = 0.003) or IgG (r = 0.54, P = 0.006) antibodies from the typhoid patients. There was no correlation with the Widal test. The TUBEX test, if performed on slides (instead of tubes) or with soluble antigen (instead of antigen-conjugated magnetic beads), suffered significantly in sensitivity. Direct agglutination tests using LPS-conjugated indicator particles performed either on slides or in microwells also failed to detect antibodies from the majority of typhoid patients. Thus, TUBEX appears to be well designed and well suited for use in the laboratory or by the bedside as a simple, rapid aid to the routine diagnosis of typhoid fever.     

The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

The human-restricted bacterial pathogen Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide and represents a significant global public health threat (1, 2). N. meningitidis is carried asymptomatically in the protective mucus layer of the throat for 5 to 25% of the population (3–5). In some cases, N. meningitidis disseminates into the bloodstream, an environment to which this bacterium is remarkably well adapted. Meningococci possess a polysaccharide capsule that protects them against complement deposition plus several membrane associated factors that are important for survival, including factor H binding protein and iron uptake systems (6). Critical to N. meningitidis survival in the bloodstream are the type IV pili (T4P), which mediate vascular colonization; nonpiliated meningococci are rapidly cleared from the blood (7–9). T4P are long filamentous appendages displayed peritrichously on the bacterium. The major pilin protein, PilE, is the primary building block of the pilus. This and other surface-displayed N. meningitidis proteins undergo antigenic variation, allowing this pathogen to evade a protective immune response (10–12).T4P are responsible for acute colonization of human blood vessels and are thus essential in establishing invasive meningococcal diseases (7, 9, 13, 14). T4P are helical polymers of the major pilin assembled by the T4P machinery (15, 16). The conserved N terminus of the major pilin is a hydrophobic α-helix that tethers the C-terminal globular domain in the inner membrane prior to pilus assembly and forms a helical array in the core of the intact pilus, displaying the globular domain on the filament surface. Pilus assembly is initiated by a cluster of pilin-like proteins called minor pilins (17–20). These “core” minor pilins are thought to localize to the pilus tip. The major pilin, PilE, is highly conserved in amino acid sequence and structure between N. meningitidis and the urogenital pathogen Neisseria gonorrhoeae with the exception of a hypervariable β-hairpin near the C terminus that is prominent on the pilus surface (21–24). In N. meningitis, PilE has been shown to bind to sialylated N-glycans on the human endothelial cell receptor CD147 (also called EMMPRIN or Basigin) (25) and the β2-adrenergic receptor (26), two membrane proteins that form a heterotrimeric complex with cytoplasmic α-actinin 4 (27).The pathogenic Neisseria possess a set of core minor pilins, PilH (FimT), PilI (PilV), PilJ (PilW), PilK, and PilX, that are encoded within a single gene cluster and prime pilus assembly (28), plus “noncore” minor pilins PilV and ComP, which are encoded elsewhere on the genome. ComP shares the canonical T4P–pilin structure of the major pilin, PilE, with the N-terminal α-helix and C-terminal globular domain (29). ComP is involved in natural transformation of exogenous DNA (30). PilV, which is highly conserved in N. meningitidis isolates (31), participates in adhesion and signaling in host cells (13, 26, 32–34). In N. meningitidis, PilV, like PilE, directly interacts with CD147 and the β2-adrenergic receptor, suggesting that PilV colocalizes with PilE within the T4P filament (13, 26). However, another report concluded that PilV functions exclusively from within the periplasm, fine-tuning pilus surface display to regulate its interactions with host cells (35).The T4P–receptor interaction represents a key step in N. meningitidis adhesion and colonization of endothelial cells in peripheral and brain vasculature and is thus an attractive target for preventive and therapeutic approaches to tackle meningococcal infection. Interfering with piliation prevents N. meningitidis colonization of human endothelial cells and vasculature (36) and improves sepsis outcome in a mouse model grafted with human skin (9). Although both PilE and PilV are involved in adhesion, PilE exhibits considerable amino acid sequence variability in its exposed hypervariable region. This variability contributes to Neisseria immune escape. In contrast, PilV is highly conserved and has been shown to be immunogenic in humans (37). Thus, PilV may prove to be a more promising target than PilE for blocking endothelial cell adhesion. A molecular understanding of this minor pilin with respect to its structure, localization within the pilus, and interactions with host receptors will be valuable in assessing its potential as a therapeutic target. Here, we report the atomic structure of PilV and superresolution microscopy images showing that it is incorporated throughout the N. meningitidis T4P. We identify residues involved in adhesion to host cells and map these onto the PilV structure, modeled within the cryoelectron microscopy (cryoEM)-derived pilus filament structure. Finally, we show that anti-PilV antibodies inhibit meningococcal adhesion in vivo. These data provide insights into PilV-mediated adhesion and suggest that blocking its adherence functions may inhibit N. meningitidis vascular colonization and pathogenesis.