Glenn J. Merkel

Histologic analysis of neural elements in the human sacroiliac joint

Study Design. The posterior ligament of the human sacroiliac joint was examined for nerves and nerve endings using histologic and immunohistochemical techniques. Objective. To identify nerve fibers and mechanoreceptors in the posterior ligament. Summary of Background Data. According to the findings of previous studies, the human sacroiliac joint receives myelinated and unmyelinated axons that presumably conduct pain and proprioceptive impulses derived from mechanoreceptors and free nerve endings in the human sacroiliac joint. Methods. Tissue obtained from six patients was stained with gold chloride and that obtained from six additional patients was stained using antibodies specific for substance P and protein gene product 9.5. Results. The staining of joint tissue using the gold chloride technique showed myelinated and unmyelinated nerve fibers, two morphotypes of paciniform encapsulated mechanoreceptors, and a single nonpaciniform mechanoreceptor. Analysis using immunohistochemical staining for protein gene product 9.5 did not unequivocally show axons, nerve fascicles, or mechanoreceptors. Similarly, analysis based on immunohistochemical staining for substance P, one of several neurotransmitters known to signal pain from the periphery, showed reactive elements that may have been nerves, but because of background staining, could not be positively identified as such. Conclusions. The presence of nerve fibers and mechanoreceptors in the sacroiliac ligament demonstrates that the central nervous system receives information, certainly proprioceptive, and possibly pain from the sacroiliac joint. Although it is not known how the central nervous system uses such information, it seems reasonable to speculate that the proprioceptive information is used to optimize upper body balance at this joint. In addition, because the staining techniques used generally to show nerves and nerve elements in periarticular connective tissue are nonspecific, the distinction between neural and nonneural should be made on the basis of both morphologic and staining characteristics.

Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells.

BackgroundProbiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD). Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI), is potentially deadly. C. difficile associated diarrhea (CDAD) is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892) on C. difficile-mediated cytotoxicity using Caco-2 cells as a model.MethodsExperiments were carried out to test if the cytotoxicity induced by C. difficile- conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS) from LDB B-30892 in different dilutions (1:2 to 1:2048). In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile.Results and discussionCo-culturing of LDB B-30892 with C. difficile inhibited the C. difficile- mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16) on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin preparation (CFT) of separately cultured C. difficile, the LDB B-30892 CFS was inhibitory to C. difficile CFT-mediated cytotoxicity at a ratio of 1:8 (LDB B-30892 CFS:C. difficile CFT). We failed to find any similar inhibition of C. difficile- mediated cytotoxicity when other probiotic organisms were tested in parallel to LDB B-30892. Our data of cytotoxicity experiments suggest that LDB B-30892 releases one or more bioactive component(s) into the CFS, which neutralizes the cytotoxicity induced by C. difficile, probably by inactivating its toxin(s). Our data also indicate that CFS from LDB B-30892 reduced the adhesion of C. difficile by 81%, which is significantly (P <0.01) higher than all other probiotic organisms tested in this study.ConclusionThis study reveals the very first findings that Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892) can eliminate C. difficile-mediated cytotoxicity, using Caco-2 cells as a model. The study also demonstrates that LDB B-30892 can reduce the colonization of C. difficile cells in colorectal cells. More study is warranted to elucidate the specific mechanism of action of such reduction of cytotoxicity and colonization.

Isolation and Peptidoglycan of Gram-negative Hydrocarbon-utilizing Thermophilic Bacteria

SUMMARY: Four Gram-negative, non-sporulating, aerobic, obligate thermophilic bacteria, isolated from non-thermal environments by enrichment with n-heptadecane as substrate, utilized n-alkanes, carbohydrates and organic acids as sole source of carbon and energy and also grew on complex media. The growth rate of these organisms, when utilizing n-heptadecane as substrate, was markedly increased by adding a low concentration (7·5 mg 1-1) of yeast extract. They grew optimally between 55 and 65 °C, and at a pH between 6·2 and 7·5. The mol% G + C for all was between 51 and 58. On the basis of the amino acid and amino sugar compositions of their peptidoglycan, these organisms and other Gram-negative thermophilic bacteria can be divided into four distinct groups. Group A includes the newly isolated hydrocarbon-utilizing bacteria which have nearly equimolar amounts of glutamic acid, alanine, diaminopimelic acid and glucosamine. Group B consists of obligate hydrocarbon-utilizing microbes that have lower molar ratios of glutamic acid and diaminopimelic acid, and contain either ornithine or lysine. The previously isolated non-hydrocarbon-utilizing thermophiles (k-2, Thermus aquaticus YT-1, Thermus x-1) and a newly isolated organism from a hot spring comprise group C and contain glycine, ornithine, no diaminopimelic acid, and much lower molar ratios of glutamic and muramic acids than in groups A and B. Thermomicrobium roseum lacked peptidoglycan and is placed separately in group D.

Impaired clearance and organ localization of Candida albicans in obstructive jaundice

Sepsis is a major cause of morbidity and mortality in infants with cholestatic jaundice. This may be attributed to altered host defense mechanisms. Fungal infection frequently occurs in immunocompromised patients. This study evaluates the effect of biliary obstruction on blood clearance and organ localization of radiolabeled viable Candida albicans. Male Sprague-Dawley rats (140 to 150 g) were placed in 2 groups. Group I (n = 30) were sham-operated controls. Group II (n = 90) underwent ligation and division of the distal common bile duct (CDL). At 1, 2, and 3 weeks following CDL, 10(7) cells/mL radiolabeled viable C albicans were injected via the tail vein. The final distribution of the organisms was calculated and expressed as the mean percent of radiolabeled organisms per gram and per total organ. Blood clearance of C albicans was similarly rapid in both groups. However, there was a significant decrease in the trapping of fungi by the rat liver Kupffer cells (20.3% +/- 7.9% v control 42.5% +/- 15%; P greater than .001), and increased pulmonary localization of bacteria 3 weeks following CDL (53.6% +/- 13.2% v control 41.4% +/- 6.4%). The significant decrease in liver trapping and increased lung localization of C albicans in CDL rats, may result in systemic reemergence of fungi and play a role in the susceptibility to fungal infection in jaundiced subjects.

Blood clearance and organ localization of Candida albicans and E coli following dual infection in rats

Immunosuppressed prematures, cancer patients, and transplant recipients are susceptible to bacterial or fungal sepsis or both. This report evaluates whether the ability of the reticuloendothelial system (RES) to remove blood-borne viable radiolabeled 35S Escherichia coli and 3H-Leucine Candida albicans is adversely affected by a dual intravenous challenge of these organisms. Male Sprague Dawley rats (n = 150) weighing 175 to 180 g were placed in 5 experimental groups (n = 30). Group I received intravenous (IV) C albicans (10(7)/mL), group II received E coli (10(9)/mL), group III received a dual injection of C albicans and E coli, group IV received Candida 1 hour prior to E coli, and group V received E coli 1 hour prior to fungi. At 1, 4, and 24 hours, tissue samples (50 to 100 mg) of liver, spleen, kidneys, and lungs were processed for liquid scintillation counting. Organ distribution of bacteria and fungi was calculated and expressed as mean percent +/- SD of labeled organisms. The liver trapped 72% +/- 10% and the lungs 1.1% +/- 0.3% of E coli (group II) (P < .001). The organ distribution of Candida (group I), however, was similar in liver and lungs (42.5% +/- 10% and 41.4% +/- 6.4%, respectively). Liver localization of E coli was unaffected by simultaneous or staggered fungal injection (groups III, 4, and V). Lung distribution of E coli following dual injection (group III) was significantly higher than controls (group II) (3.6% +/- 0.7% v 1.1% +/- 0.3%; P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of Staphylococcus epidermidis with endothelial cells in vitro

Abstract Staphylococcus epidermidis is a leading cause of nosocomial bacteremia, yet virtually nothing is known about how this pathogen interacts with human endothelial cells. We present evidence here that two biofilm-producing strains of S. epidermidis adhere to two types of endothelial cell lines in vitro and that adherence is significantly increased after briefly heat-treating the bacteria at 40°C in the presence of calcium. This mild heat treatment resulted in bacteria that were 5 to more than 20 times more adherent than untreated controls. While the adherence of bacteria in all phases of growth was increased after heat treatment, heat-treated late stationary phase cells were generally the most adherent. Electron microscopy demonstrated that S. epidermidis was internalized and appeared to exist free in the cytoplasm. Adherence to endothelium, should it occur in vivo during bacteremia, may be a virulence factor associated with this bacteriums pathogenesis.

Characterization of a monoclonal antibody that binds to an epitope on soluble bacterial peptidoglycan fragments.

ABSTRACT We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from Staphylococcus aureus and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.

Mitogenic and morphogenic effects of a bovine salivary gland extract on astrocytes and fibroblasts

The presence of mitogenic and morphogenic activity in extracts of bovine salivary (parotid) glands is reported. The crude and partially purified extracts stimulated cultured rat cerebellar cells (astrocytes) and skin fibroblasts to undergo morphogenesis. The incorporation of [3H]thymidine was also stimulated in astrocytes, skin fibroblasts, and established fibroblastic cell lines. Growth-promoting activity was also demonstrated. The expression of maximum mitogenic activity in skin fibroblast cultures, but not kidney fibroblast cultures, required the presence of serum. The biological activity had an apparent native molecular weight greater than 230,000, was heat-sensitive, trypsin-resistant, and slightly sensitive to the action of papain.

Phospholipid composition and heat sensitivity in a thermophilic bacterium

A Gram-negative thermophilic bacterium designated strain LEH-1, survived under non-growth conditions, temperatures 5 degrees C above its maximum for growth when acetate or n-heptadecane served as carbon source, whereas cells grown with glucose or glycerol were markedly more sensitive to elevated temperatures. The total extractable lipids from strain LEH-1 and the concentrations of phosphatidylethanolamine and phosphatidic acid in the cytoplasmic membranes was altered by the growth substrate. Growth with acetate of n-heptadecane as carbon source yielded cells containing more phosphatidic acid than phosphatidylethanolamine. Conversely, more phosphatidylethanolamine was present in cells following growth with glucose or glycerol as carbon source. The relative amount of specific fatty acids and distribution in major phospholipid moieties was also affected by the growth substrate. These differences in composition may reflect an alteration in the physical properties of the cellular membrane and account for the divergence in heat sensitivity.