A new protocol for multiple inhalation of IFN-gamma successfully treats MDR-TB: a case study.

SETTING: Aerosolized interferon-gamma (IFN-gamma) leads to transient conversion of sputum smears in multidrug-resistant pulmonary tuberculosis (MDR-TB). OBJECTIVE: To test long-term conversion of sputum smears using the new Jena protocol. DESIGN: Four MDR-TB patients were treated with aerosolized recombinant IFN-gamma (rIFN-gamma) twice weekly for 8 weeks and anti-tuberculosis drugs. Patients were monitored clinically and T-cell subpopulations were analyzed. RESULTS: The treatment was well tolerated. All sputum smears cleared within 6-8 weeks, and radiological signs of recovery lasted in all patients for 73-106 months (the entire follow-up period). Before treatment, a patient with a 20+ year history of TB showed no gammadelta T-cells; these cells appeared during treatment. The proportion of natural killer (NK) cells was enhanced during treatment and remained elevated. The proportion of CD4+/CD25+ T-cells in the blood rose after treatment and remained elevated at 2 and 10 months afterwards. No significant change in T-cell levels appeared in patients with a shorter history of TB, except for a tendency toward a slight increase in gammadelta T-cells during treatment. CONCLUSION: We invite further confirmation, but aerosolized rIFN-gamma plus anti-microbial treatment cured MDR-TB in this case study. The optimal dosing schedule needs to be determined.

Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils.

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO.

Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis.

We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.

Avulsion of the bovine prepuce from its attachment to the penile integument during semen collection with an artificial vagina.

Avulsion of the prepuce (AP) from the penile integument during intromission into an artificial vagina (AV) is the most common injury involving the penis and prepuce of bulls collected for the production of frozen semen at artificial insemination (AI) centers. We observed clinical cases at two AI centers. Because temporary loss of seminal collections from genetically superior bulls occurs and the possibility of impotency exists, the economical loss from AP is significant. Surgery, rather than a nonsurgical procedure, is the preferred method for repair of AP if the diagnosis occurs on the same day as the injury. After injury, seminal collection is avoided. The bull can then begin a conditioning program prior to returning to a normal semen collection schedule. A specific etiology for AP remains undetermined; however, many variables potentially involved in its occurrence are known. Bulls with good libido following 4 to 5 yr of sexual inactivity are particularly susceptible to AP. No genetic predisposition was observed. At present, we believe that AP is caused by many factors and that implementing preventive measures does help.

Surgical approach for pinealectomy in the calf.

Pinealectomy was performed on 7 calves, and a sham operation was performed on 3 calves. Two pinealectomized calves died immediately. The 5 remaining pinealectomized and 3 sham-operated control calves are presently involved in experimental studies. The surgical technique for exposure of the pineal gland was a rectangular (rostrotentorial) craniectomy between the median line and the horn bud. Removal of portions of the frontal and parietal bones, measuring 6 cm by 4 cm, provided adequate exposure. Positioning the head was important in order to gain the best access to the pineal gland. Water deprivation, hypertonic solution, elevation of the head, and hyperventilation prevented brain edema during the operation. The dura mater was sutured and glucocorticoids were administered to prevent postsurgical brain edema. Bone graft or plate was not placed over the craniectomy site. The soft tissue flap was repositioned over the dura mater and sutured in place--the periosteum and muscle comprising 1 layer and the skin a 2nd layer. Postsurgical complications were observed only in the 2 calves which died and were the result of hemorrhage and leukomalacia.

Efficacy of amoxicillin trihydrate for the treatment of experimentally induced foot rot in cattle.

Twenty holstein heifers were intradermally inoculated in the interdigital skin with a suspension containing Fusobacterium necrophorum and Bacteroides melaninogenicus to induce acute foot rot. Lesions, lameness, and swelling were evaluated during the study, using a subjective scoring system. Rectal temperature, species and number of bacteria isolated, and change in body weight were monitored throughout the study. Ten heifers (treated) were given amoxicillin trihydrate (10 mg/kg of body weight, IM) for 5 days, beginning at the onset of lameness. The remaining 10 heifers (controls) were given physiologic saline solution IM. Treated heifers had less severe lesions and greater weight gain than did control heifers. Rectal temperatures of treated heifers did not differ significantly from those of control heifers. It was concluded that administration of amoxicillin trihydrate early in the course of acute foot rot may reduce the severity of lesions associated with foot rot in cattle.

Use of an enzyme-linked immunosorbent assay to estimate prevalence of paratuberculosis in cattle of Florida.

A survey was conducted from 1986 through 1987, for which an ELISA was used to obtain information on the prevalence of Mycobacterium paratuberculosis infection in cattle of Florida. Results revealed prevalence of 8.6% in beef cattle and 17.1% in dairy cattle. In beef and dairy cattle, prevalence increased with increasing herd size. It was concluded that ELISA-detectable circulating antibodies to M paratuberculosis are widespread in cattle of Florida.

Diversity of Salmonella serotypes in cull (market) dairy cows at slaughter.

OBJECTIVE: To determine the diversity of Salmonella serotypes isolated from a large population of cull (market) dairy cows at slaughter. DESIGN: Cross-sectional study. SAMPLE POPULATION: Salmonella organisms isolated from the cecal-colon contents of 5,087 market dairy cows. PROCEDURE: During winter and summer 1996, cecal-colon contents of cull dairy cows at slaughter were obtained from 5 US slaughter establishments. Specimens were subjected to microbiologic culturing for Salmonella spp at 1 laboratory. Identified isolates were compared with Salmonella isolation lists published by the Centers for Disease Control and Prevention (CDC) and the National Veterinary Services Laboratory (NVSL) for approximately the same period. The Simpson diversity index was used to calculate the likelihood that Salmonella isolates selected randomly by establishment were different. RESULTS: Of 58 Salmonella serotypes identified, Salmonella ser. Montevideo was the most prevalent. Two of the top 10 CDC serotypes identified from in 1996, Salmonella ser. Typhimurium and S Montevideo, appeared on our top 10 list; 8 of the top 10 were found on NVSL listings. Thirty-one of 59 S. Typhimurium isolates were identified as DT104 and found at a west slaughter establishment, 30 during the winter and 1 during the summer. The greatest diversity of serotypes was at a southeast establishment during the summer; the least diversity was at a central establishment in the winter. CONCLUSIONS AND CLINICAL RELEVANCE: 58 Salmonella serotypes were isolated from market dairy cows at slaughter and could pose a threat for food-borne illness. Salmonella Montevideo was the most frequently isolated serotype and may contribute substantially to salmonellosis in dairy cattle.

Prevalence of Salmonella spp in cull (market) dairy cows at slaughter.

OBJECTIVE: To determine the prevalence of Salmonella spp in the cecal-colon contents of cull (market) dairy cows at slaughter because of potential public health ramifications. DESIGN: Survey study. SAMPLE POPULATION: Cecal-colon contents collected from 5,087 cull (market) dairy cows at slaughter at 5 slaughter establishments across the United States. PROCEDURE: During 2 periods of the year, winter (January and February) and summer (July through September), 5 cull (market) cow slaughter establishments in the United States--west (WE), southeast (SEE), central (CE), north central (NCE), and south central (SCE)--establishments were visited, and cecal-colon contents of cull dairy cows were obtained at the time of slaughter. Samples were examined by microbiologic culture at a single laboratory for Salmonella spp. RESULTS: Salmonella spp were detected in 23.1% of cecal-colon content samples from cull dairy cows across the 5 slaughter establishments. The highest site prevalence (54.5%) was detected at the WE during the summer period, whereas the lowest was found at the CE during the summer (4.3%) and at the NCE during the winter (4.5%). Considerable variation in the daily prevalence of Salmonella spp was found, particularly at the WE and the SCE. Salmonella spp were isolated from 93% of cecal-colon contents collected on a summer day at the WE. CONCLUSIONS AND CLINICAL RELEVANCE: Results strongly suggest that there is a high prevalence of Salmonella spp in cull dairy cows at slaughter, which could burden Hazard Analysis Critical Control Point programs implemented in slaughter establishments. Procedures to reduce Salmonella load at the dairy farm and during transport to slaughter could reduce the risk of spread during the slaughter process.

Filter selection for five color flow cytometric analysis with a single laser.

Flow cytometry has evolved from single- and two-color analysis to the current use of 11-16 colors. The relatively bright excitation spectra of most fluorochromes have made color compensation a challenge especially when performed manually. We describe how by choosing filters with narrower bandwidths results in the color compensation values between FITC, PE, PE-TxR (ECD), PE-Cy5, and PE-Cy7 that range from 0 % to 50% depending on the combination of fluorochromes. Peripheral blood mononuclear cells were stained with alpha-CD4-FITC, alpha-CD27-PE, alpha-CD62L-ECD, alpha-CD45RA-PE-Cy5 and alpha-CD3-PE-Cy7. The samples were acquired on a MO Flo. The initial (first) and second filter sets for our experiments consisted of 530/30 or 519/20 for FITC, 580/30 or 575/20for PE, 630/30 or 630/22 for PE-TxR (ECD), 670/30 or 675/20 for PE-Cy5 and 740LP or 780/40 for PE-Cy7. Nonstained cells were used to adjust the threshold values of detection for each photo multiplier tube (PMT) for each filter set. The mean fluorescent intensity (MFI) of each fluorochrome was not reduced to any great extent by either filter set. However, the compensation value between PE and PE-TxR (ECD) with the first filter selection ranged from 84% to 89% and with the second set of filters it was 25-36%. In addition, the compensation between PE-TxR (ECD) and PE-Cy5 were reduced to 30.2% from 44.2% with the second filter set. The reduction of filter bandwidths that results in minimizing spectral overlaps without lost of signal provides a method by which discrimination of signals between PE containing fluorochromes can be achieved.

[Respiratory failure and pulmonary fibrosis as a late side-effect after chemotherapy-induced by oxygen administration]. / Respiratorische Insuffizienz und pulmonale Fibrose als Spätfolge einer Chemotherapie-induziert durch Sauerstoffexposition.

Pulmonary fibrosis (PF) may develop following successful chemotherapy for malignancy, even if such therapy is not combined with radiotherapy. Bleomycin, which is known to induce acute pneumonitis and lung fibrosis, is especially associated with chemotherapy-induced PF, and bleomycin-induced pulmonary fibrosis can occur more than five years after such therapy. Additionally, supplemental oxygen therapy can trigger the onset of pneumonitis and lethal PF in patients who have previously received bleomycin therapy. Careful assessment of lung function via spiroergometry and arterial blood gas analysis during exercise are required if the administration of supplemental oxygen is considered. Two case reports reveal the potential lethal risk of oxygen for patients who have been treated with bleomycin: (1) a patient with successfully resected and treated basal tongue carcinoma and (2) a patient in remission after being treated for non-Hodgkin lymphoma. Single and double lung transplantation is the only therapeutic option for patients with severe, oxygen-induced PF and should be included as an indication for lung transplantation. Early recognition of pulmonary diffusion abnormalities and establishing a risk profile, as well as consequent monitoring of pulmonary function, may help to avoid or at least reduce the risk of PF induced by oxygen therapy when administered to patients who have previously been given bleomycin.

[Molecular mechanisms of signal transduction in the framework of CD95 and CD65 receptor-induced apoptosis of human eosinophilic granulocytes. Effect on GM-CSF mediated viability and relationship to intracellular bcl-2 expression]. / Molekularer Mechanismus der Signaltransduktion im Rahmen der CD95- und CD69-Rezeptor-induzierten Apoptose humaner eosinophiler Granulozyten. Einfluss auf die GM-CSF-vermittelte Viabilität und Beziehung zur intrazellulären bcl-2-Expression.

Infiltration of eosinophils into the airways plays a central role in the pathophysiology of asthma. Human blood eosinophils express apoptosis-inducing receptors (e.g. CD95R and CD69R) regulating both viability and survival and, thus, the extent of eosinophil infiltration into the airways. Signal transduction processes induced by occupation of the CD69 receptor expressed by eosinophils are insufficiently known. Purified human peripheral blood eosinophils (MACS, purity > 99%) were pre-incubated with a GM-CSF for 18 h and stimulated with alpha-CD69mAb (clon TP1/55), alpha-CD95mAb (clon CH-11), and as a control alpha-CD11bmAb (clon Bear-1). The specificity of receptor ligation was assessed using a blocking mAb (Klon ZB4). Phenotype, viability, apoptosis and bcl-2-expression were measured employing flow cytometry. alpha-CD95mAb (1 microgram/ml) induced apoptosis both in control and GM-CSF (10 ng/ml) treated eosinophils. Similarly, alpha-CD69mAb (10 micrograms/ml) induced apoptosis of GM-CSF-stimulated CD69+ cells after an incubation period of 114 h which was not affected by a CD95 blocking mAb. Naive eosinophils showed a basale, bcl-2-expression, which decreased to 30% after 66 h. In the presence of GM-CSF, intracellular bcl-2-concentration remained unchanged. Following stimulation with alpha-CD69mAb or alpha-CD95mAb, a dose-dependent decline of the bcl-2-expression was detected, whereas alpha-CD11bmAb (10 micrograms/ml) had no effect. The data suggest that both CD95R- and CD69R-induced apoptosis of human eosinophils involves a bcl-2-dependent signal transduction mechanism.

Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.