Effects of Purified Staphylococcal Alpha Toxin on the Ultrastructure of Human and Rabbit Erythrocytes

Previous studies have suggested that the primary site of action of purified staphylococcal alpha toxin is the cell membrane. Scanning and transmission electron microscopy studies were undertaken, therefore, to define toxin-induced alterations in the surface morphology of rabbit and human red blood cells. During the prelytic lag phase, scanning electron microscopy revealed multiple discrete blisters on the surface of rabbit red blood cells; during hemolysis, cellular collapse and ghosts were seen, but most striking was the separation of large fragments of cell membrane from red blood cell surfaces. In contrast, alterations in less sensitive human red blood cells were limited to occasional fingerlike protrusions during the period of accelerated lysis. Transmission electron microscopy substantiated these changes. These studies have provided further evidence that the cell membrane is the primary site of action of staphylococcal alpha toxin.     

Complement studies in membranoproliferative glomerulonephritis

Detailed studies of the complement system were carried out in fifteen patients with membranoproliferative glomerulonephritis. The findings of reduced levels of C3 and C7 and of circulating breakdown products of C3 in fresh plasma suggested in vivo complement activation. Low C3 levels were associated with the presence of a serum factor (the C3 nephritic factor C3NeF) which was capable of breaking down C3 in normal serum in vitro. Metabolic studies using radioactive iodine labelled C3 showed no evidence of accelerated in vivo breakdown of parenterally administered C3 suggesting that hypocomplementaemia is either maintained by diminished C3 synthesis or that accelerated catabolism is occurring in a pool that does not freely exchange with parenterally given C3. The C3 nephritic factor has so far only been identified in patients with membranoproliferative nephritis and is therefore of major diagnostic significance in patients with glomerular disease.     

Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.     

Controlled comparison of BacT/ALERT FAN aerobic medium and BATEC fungal blood culture medium for detection of fungemia

Yeasts are an increasingly common cause of nosocomial bloodstream infections. Methods for their detection are many; controlled comparisons are few. The vented FAN aerobic blood culture medium has been shown to be superior to the standard BacT/ALERT aerobic medium for the detection of fungemia as well as bacteremia. The BACTEC selective fungal medium (FM) (BD Biosciences, Sparks, Md.) allowed detection of more episodes of fungemia than did a resin-containing medium with equal volumes of blood cultured. Therefore, we compared vented FAN to FM for the ability to recover fungi from the blood of patients who were at increased risk of having fungemia. From 5,109 cultures processed for which both FAN and FM bottles were adequately filled, fungi were recovered from 87 cultures. Of these, 47 were detected with both bottles, 12 were detected with FAN only, and 28 were detected with FM only (P < 0.05). FAN was the first bottle positive for 36 of the 47 cultures for which both bottles yielded the same fungus, whereas the FM bottle was the first bottle positive for 11 cultures (P < 0.001). A total of 54 episodes of fungemia were identified, with 40 detected by both media, 4 detected only by FAN, and 10 detected only by FM (P value, not significant). We conclude that the vented FAN aerobic bottle is comparable to the FM bottle for detection of episodes of yeast infection but has the added benefit of detecting bacteria.     

Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia.

FAN medium was formulated to improve microbial recovery, particularly for fastidious microorganisms and for microorganisms causing sepsis in patients receiving antimicrobial therapy. In a controlled clinical evaluation performed at four university-affiliated hospitals, FAN anaerobic bottles were compared with standard anaerobic bottles for yield, speed of detection of microbial growth, and detection of septic episodes. A total of 10,431 blood culture sets were received; both anaerobic bottles of 7,694 blood culture sets were adequately filled with blood. Altogether, 925 isolates were recovered: 557 that were the cause of sepsis, 99 that were indeterminate as the cause of sepsis, and 269 contaminants. More Staphylococcus aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Escherichia coli (P < 0.02), and all microorganisms combined (P < 0.005) were recovered from FAN bottles; more nonfermentative gram-negative bacilli (P < 0.05), Torulopsis glabrata (P < 0.001), and other yeasts (P < 0.01) were recovered from standard bottles. Growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Enterococcus faecalis (P < 0.025), streptococci other than Streptococcus pneumoniae (P < 0.01), and all microorganisms combined (P < 0.001) was detected earlier in standard bottles; growth of more isolates of E. coli (P < 0.05) and anaerobic bacteria (P < 0.01) was detected earlier in FAN bottles. The mean times to detection were 14.2 and 16.1 h for standard and FAN bottles, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathology of the Glomerulus in Sickle Cell Anemia With and Without Nephrotic Syndrome

Glomeruli from 6 cases of sickle cell disease (SS) with the nephrotic syndrome (NS) were compared histologically and quantitatively with glomeruli from 9 cases of SS, 10 cases of sickle cell trait (SCT), 4 cases of other hemoglobinopathies, all without NS, and normal controls. Five of 6 patients with SS and NS had extensive reduplication of their glomerular basement membranes and mild mesangial proliferation. Similar but milder lesions occurred in SS without NS but not in SCT or controls. Incidental renal disease occurred in 1 patient with SS and NS. Nephrotic syndrome was probably secondary to effects of sickle cell disease. Glomeruli in SS were significantly larger (>70%) than in SCT and controls. Mean total glomerular area per unit area of cortex in SS with normal BUN significantly exceeded that of SCT, which, in turn, was significantly greater than that of controls. Mechanisms for the histologic lesions and hypertrophy of the glomeruli were suggested.     

Is fecundability associated with month of birth? An analysis of 19th and early 20th century family reconstitution data from The Netherlands

The relationship between fecundability and month of birth was investigated in a cohort of 1526 women who married between 1802 and 1929, using only women whose first marriage occurred before the age of 35 years. On the basis of their time to pregnancy (TTP, calculated as time between wedding and first birth minus gestational length), women were categorized into two groups: fecunds (TTP up to 12 months or prenuptial conceptions, n = 1348) and subfecunds (TTP >18 months, n = 118). By use of logistic regression, cosinor functions with a period of 1 year or 6 months and variable shift and amplitude were fitted through the monthly odds of subfecunds versus fecunds. The best fitting curve was unimodal, with a zenith in September (P = 0.13 for H0: no differences). Exclusion of childless women (n = 36, minimum follow-up 5 years) from the subfecunds led to a similar curve (P < 0.01), while childless women, as compared with fecunds, showed a birth distribution that was best represented with a bimodal curve with zeniths in January and July (P = 0.06). This study provides evidence for the existence of differences in fecundability by month of birth. The cause of this relationship is unclear, but may lie in a melatonin-dependent circannual variability of the quality of the oocyte.      

Identification and localization of G protein subunits in human spermatozoa

Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility.     

Clinical importance of increased sensitivity of BacT/Alert FAN aerobic and anaerobic blood culture bottles.

Two recent multicenter blood culture studies found that BacT/Alert FAN (FAN) bottles (Organon Teknika, Durham, N.C.) had increased yields in detecting bacteremia and fungemia compared with standard BacT/Alert (STD) bottles. Because the clinical importance of this increase in microbial recovery is unknown, we performed a retrospective analysis to determine the frequency with which FAN bottles were the sole means of detecting an episode of bacteremia. There were 1,047 positive blood cultures in which both study bottles were adequately filled and the organism isolated was judged to be the cause of sepsis: 240 (23%) were positive only in FAN bottles and 73 (7%) were positive only in STD bottles. Of a total of 664 episodes of bacteremia, 126 (19%) were identified only by FAN bottles and 43 (7%) were identified only by STD bottles (P < 0.0001). Episodes detected only by FAN bottles more often were recurrent events (23 of 126, or 18%) than episodes detected only by STD bottles (2 of 43, or 5%) (P < 0.05) and more commonly occurred in patients receiving theoretically effective antibiotic therapy (33 of 126 [26%] versus 4 of 43 [9%]) (P < 0.05). The medical records for patients with 127 of these episodes (92 FAN bottles only; 35 STD bottles only) were available for review. More than half of both FAN bottle-only (60 of 92, or 65%) and STD bottle-only (20 of 35, or 57%) episodes were judged to be clinically important. We conclude that FAN bottles improve the detection of bacteremia and that the majority of the additional episodes detected are clinically important. The benefits of the greater yield in specific patient populations must be balanced against the higher costs of FAN bottles.