The release of pituitary LH and sprouting of LH-releasing hormone (LHRH)-containing neurons after anterior hypothalamic deafferentation

The chronology of changes in plasma luteinizing hormone (LH) and the distribution of immunoreactive neuronal processes containing LH releasing hormone (LHRH-ir) were studied in the female rat after surgical interruption of anterior neural connections of the mediobasal hypothalamus (MBH). Spontaneous LH surges on the afternoon of proestrus and LH release after estradiol benzoate (EB) followed 48 h later by progesterone (P) administration were studied in ovariectomized (OVX) rats. The maximum increase in plasma concentrations (delta maxLH) after EBP was calculated for each rat at several intervals over 140 days. Control animals given EBP at monthly intervals after OVX had comparably large delta maxLH surges during the first few months of study. However, a gradual decline in control delta maxLH followed becoming significant 3 months after the start of the experiment. In contrast, frontal cuts (FC), which interrupted anterior MBH connections, produced an abrupt decrease in delta maxLH surges after EBP to 11% of preoperative levels. However, during subsequent EBP trials, there was a gradual improvement in LH surges to about 50% of preoperative levels over 100 days. In some cases, individual improvement became equal to preoperative LH surge levels, in others there was no recovery. Examination of LHRH-ir nerve fiber growth responses after FC suggested that sprouting by these peptide-containing neuronal processes may have contributed to the functional improvements observed.     

Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A.

Monoclonal antibody, affinity-purified antibody, and monospecific antiserum against toxin A were produced. The monoclonal antibody was an immunoglobulin G2a kappa chain isotype that immunoprecipitated toxin A, as shown by crossed immunoelectrophoresis. These antibodies were compared by counterimmunoelectrophoresis, latex agglutination, and indirect enzyme-linked immunosorbent assay for their sensitivity in detecting toxin A. Our findings indicate that these antibodies may be useful as immunodiagnostic reagents for Clostridium difficile disease.     

Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies.

Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.     

In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods.

We evaluated test discriminatory power and DNA type alterations among methicillin-resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality.     

Molecular characterization of the Clostridium difficile toxin A gene

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.     

Comparison of Two Culture Methods for Detection of Tobramycin-Resistant Gram-Negative Organisms in the Sputum of Patients with Cystic Fibrosis

A culture method utilizing quantitative plating on antibiotic-containing media has been proposed as a technique for the detection of tobramycin-resistant organisms that is more sensitive than standard methods. Typical sputum culture methods quantitate the relative amounts of each distinct morphotype, followed by antibiotic susceptibility testing of a single colony of each morphotype. Sputum specimens from 240 cystic fibrosis patients were homogenized, serially diluted, and processed in parallel by the standard method (MacConkey agar and OF basal medium with agar, polymyxin, bacitracin, and lactose) and by plating on antibiotic-containing media (MacConkey agar with tobramycin added at 25 microg/ml [MAC-25] and 100 microg/ml [MAC-100]). MICs of tobramycin were determined for all Pseudomonas aeruginosa isolates by broth microdilution. Growth of P. aeruginosa on MAC-25 was considered to be equivalent to a tobramycin MIC of > or = 16 microg/ml, and growth on MAC-100 was considered to be equivalent to a tobramycin MIC of > or = 128 microg/ml. Analysis of method-specific detection rates showed that tobramycin-containing medium was more sensitive than the standard method for the detection of tobramycin-resistant P. aeruginosa, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans but was less sensitive for the detection of Burkholderia cepacia than the standard method. When MICs for P. aeruginosa that grew on tobramycin-containing medium were tested by broth microdilution, the MICs for 28 of 121 strains (23%) growing on MAC-25 and 22 of 56 strains (39%) growing on MAC-100 were MICs < 16 and < 128 microg/ml, respectively. Addition of a tobramycin-containing MacConkey plate to the routine media for sputum culture may provide additional, clinically relevant microbiologic data.     

A randomized trial comparing combination electron-beam radiation and chemotherapy with topical therapy in the initial treatment of mycosis fungoides

Mycosis fungoides is a T-cell lymphoma that arises in the skin and progresses at highly variable rates. Nonradomized studies have suggested that early aggressive therapy may improve the prognosis in this usually fatal disease. We studied 103 patients with mycosis fungoides, who, after complete staging, were randomly assigned to receive either combination therapy, consisting of 3000 cGy of electron-beam radiation to the skin combined with parenteral chemotherapy with cyclophosphamide, doxorubicin, etoposide, and vincristine (n = 52) or sequential topical treatment (n = 51). The prognostic factors were well balanced in the two groups. Combined therapy produced considerable toxicity: 12 patients required hospitalization for fever and transient neutropenia, 5 had congestive heart failure, and 2 were later found to have acute nonlymphocytic leukemia. Patients receiving combined therapy had a significantly higher rate of complete response, documented by biopsy, than patients receiving conservative therapy (38 percent vs. 18 percent; P = 0.032). After a median follow-up of 75 months, however, there was no significant difference between the treatment groups in disease-free or overall survival. We conclude that early aggressive therapy with radiation and chemotherapy does not improve the prognosis for patients with mycosis fungoides as compared with conservative treatment beginning with sequential topical therapies.     

Conotruncal anomalies in the trisomy 16 mouse: an immunohistochemical analysis with emphasis on the involvement of the neural crest

The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse.     

Changes in estrogen and progestin receptor binding resulting from retrochiasmatic knife cuts

Retrochiasmatic frontolateral knife cuts (FLC) or sham operations (Sham) were performed with a Halasz-type knife. All animals were primed with estrogen plus 0.5 mg progesterone (P) and tested for lordosis both before and after surgery. Two weeks after the last test they received estradiol (E₂) in Silastic capsules and were sacrificed 2 days later for determination of either nuclear estrogen receptors or cytosol progestin receptor binding in brain and pituitary (PIT). Rats which had received FLC showed significantly lower lordosis quotients relative to Shams, and relative to their own pre-surgery scores. Nuclear E₂-receptor binding was significantly reduced in the hypothalamus (HYPO) following FLC, but not in preoptic area (POA) or PIT. No changes in cytosol P-receptor binding were observed in HYPO, POA or PIT following FLC. Our results suggest a positive correlation between the number of hypothalamic E₂-receptors and the capacity to display lordosis, and emphasize the importance of anterolateral connections to the HYPO for the progesterone-induced facilitation of lordosis.     

Reinstatement of conditioned fear in humans is context dependent and impaired in amnesia

A contextual reinstatement procedure was developed to assess the contributions of environmental cues and hippocampal function in the recovery of conditioned fear following extinction in humans. Experiment 1 showed context specificity in the recovery of extinguished skin conductance responses after presentations of an auditory unconditioned stimulus. Experiment 2 demonstrated that fear recovery did not generalize to an explicitly unpaired conditioned stimulus. Experiment 3 replicated the context dependency of fear recovery with a shock as an unconditioned stimulus. Two amnesic patients failed to recover fear responses following reinstatement in the same context, despite showing initial fear acquisition. These results extend the known functions of the human hippocampus and highlight the importance of environmental contexts in regulating the expression of latent fear associations.