Serum periostin does not reflect type 2-driven inflammation in COPD.

Although Th2 driven inflammation is present in COPD, it is not clearly elucidated which COPD patients are affected. Since periostin is associated with Th2 driven inflammation and inhaled corticosteroid (ICS)-response in asthma, it could function as a biomarker in COPD. The aim of this study was to analyze if serum periostin is elevated in COPD compared to healthy controls, if it is affected by smoking status, if it is linked to inflammatory cell counts in blood, sputum and endobronchial biopsies, and if periostin can predict ICS-response in COPD patients.Serum periostin levels were measured using Elecsys Periostin immunoassay. Correlations between periostin and inflammatory cell count in blood, sputum and endobronchial biopsies were analyzed. Additionally, the correlation between serum periostin levels and treatment responsiveness after 6 and 30 months was assessed using i.e. ΔFEV1% predicted, ΔCCQ score and ΔRV/TLC ratio. Forty-five COPD smokers, 25 COPD past-smokers, 22 healthy smokers and 23 healthy never-smokers were included. Linear regression analysis of serum periostin showed positive correlations age (B = 0.02, 95%CI 0.01-0.03) and FEV1% predicted (B = 0.01, 95%CI 0.01-0.02) in healthy smokers, but not in COPD patients In conclusion, COPD -smokers and -past-smokers have significantly higher periostin levels compared to healthy smokers, yet periostin is not suitable as a biomarker for Th2-driven inflammation or ICS-responsiveness in COPD.

Regulation of YKL-40 expression by corticosteroids: effect on pro-inflammatory macrophages in vitro and its modulation in COPD in vivo.

BACKGROUND: Macrophages constitute a heterogeneous cell population with pro- (MΦ1) and anti-inflammatory (MΦ2) cells. The soluble chitinase-like-protein YKL-40 is expressed in macrophages and various other cell types, and has been linked to a variety of inflammatory diseases, including COPD. Dexamethasone strongly reduces YKL-40 expression in peripheral blood mononuclear cells (PBMC) in vitro. We hypothesized that: a) YKL-40 is differentially expressed by MΦ1 and MΦ2, b) is decreased by corticosteroids and c) that long-term treatment with inhaled corticosteroids (ICS) affects YKL-40 levels in serum and sputum of COPD patients. METHODS: Monocytes of healthy subjects were cultured in vitro for 7 days with either GM-CSF or M-CSF (for MΦ1 and MΦ2, respectively) and stimulated for 24 h with LPS, TNFα, or oncostatin M (OSM). MΦ1 and MΦ2 differentiation was assessed by measuring secretion of IL-12p40 and IL-10, respectively. YKL-40 expression in macrophages was measured by quantitative RT-PCR (qPCR) and ELISA; serum and sputum YKL-40 levels were analyzed by ELISA. RESULTS: Pro-inflammatory MΦ1 cells secreted significantly more YKL-40 than MΦ2, which was independent of stimulation with LPS, TNFα or OSM (p < 0.001) and confirmed by qPCR. Dexamethasone dose-dependently and significantly inhibited YKL-40 protein and mRNA levels in MΦ1. Serum YKL-40 levels of COPD patients were significantly higher than sputum YKL-40 levels but were not significantly changed by ICS treatment. CONCLUSIONS: YKL-40 secretion from MΦ1 cells is higher than from MΦ2 cells and is unaffected by further stimulation with pro-inflammatory agents. Furthermore, YKL-40 release from cultured monocyte-derived macrophages is inhibited by dexamethasone especially in MΦ1, but ICS treatment did not change YKL-40 serum and sputum levels in COPD. These results indicate that YKL-40 expression could be used as a marker for MΦ1 macrophages in vitro, but not for monitoring the effect of ICS in COPD. TRIAL REGISTRATION: ClinicalTrials.gov, registration number: NCT00158847.

Dopamine in human follicular fluid is associated with cellular uptake and metabolism-dependent generation of reactive oxygen species in granulosa cells: implications for physiology and pathology.

STUDY QUESTION: Is the neurotransmitter dopamine (DA) in the human ovary involved in the generation of reactive oxygen species (ROS)? SUMMARY ANSWER: Human ovarian follicular fluid contains DA, which causes the generation of ROS in cultured human granulosa cells (GCs), and alterations of DA levels in follicular fluid and DA uptake/metabolism in GCs in patients with polycystic ovary syndrome (PCOS) are linked to increased levels of ROS. WHAT IS KNOWN ALREADY: DA is an important neurotransmitter in the brain, and the metabolism of DA results in the generation of ROS. DA was detected in human ovarian homogenates, but whether it is present in follicular fluid and plays a role in the follicle is not known. STUDY DESIGN, SIZE AND DURATION: We used human follicular fluid from patients undergoing in vitro fertilization (IVF), GCs from patients with or without PCOS and also employed mathematical modeling to investigate the presence of DA and its effects on ROS. PARTICIPANTS/MATERIALS, SETTING AND METHODS: DA in follicular fluid and GCs was determined by enzyme-linked immunosorbent assay. GC viability, apoptosis and generation of ROS were monitored in GCs upon addition of DA. Inhibitors of DA uptake and metabolism, an antioxidant and DA receptor agonists, were used to study cellular uptake and the mechanism of DA-induced ROS generation. Human GCs were examined for the presence and abundance of transcripts of the DA transporter (DAT; SLC6A3), the DA-metabolizing enzymes monoamine oxidases A/B (MAO-A/B) and catechol-O-methyltransferase and the vesicular monoamine transporter. A computational model was developed to describe and predict DA-induced ROS generation in human GCs. MAIN RESULTS AND ROLE OF CHANCE: We found DA in follicular fluid of ovulatory follicles of the human ovary and in GCs. DAT and MAO-A/B, which are expressed by GCs, are prerequisites for a DA receptor-independent generation of ROS in GCs. Blockers of DAT and MAO-A/B, as well as an antioxidant, prevented the generation of ROS (P < 0.05). Agonists of DA receptors (D1 and D2) did not induce ROS. DA, in the concentration range found in follicular fluid, did not induce apoptosis of cultured GCs. Computational modeling suggested, however, that ROS levels in GCs depend on the concentrations of DA and on the cellular uptake and metabolism. In PCOS-derived follicular fluid, the levels of DA were higher (P < 0.05) in GCs, the transcript levels of DAT and MAO-A/B in GCs were 2-fold higher (P < 0.05) and the DA-induced ROS levels were found to be more than 4-fold increased (P < 0.05) compared with non-PCOS cells. Furthermore, DA at a high concentration induced apoptosis in PCOS-derived GCs. LIMITATIONS, REASONS FOR CAUTION: While the results in IVF-derived follicular fluid and in GCs reveal for the first time the presence of DA in the human follicular compartment, functions of DA could only be studied in IVF-derived GCs, which can be viewed as a cellular model for the periovulatory follicular phase. The full functional importance of DA-induced ROS in small follicles and other compartments of the ovary, especially in PCOS samples, remains to be shown. WIDER IMPLICATIONS OF THE FINDINGS: The results identify DA as a factor in the human ovary, which, via ROS generation, could play a role in ovarian physiology and pathology. The results obtained in samples from women with PCOS suggest the involvement of DA, acting via ROS, in this condition. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from DFG MA1080/17-3 and in part MA1080/19-1. There are no competing interests.

Racial differences in healthcare utilization among patients with suspected or diagnosed preeclampsia: A retrospective cohort study.

OBJECTIVES: To analyze healthcare resource utilization and severe maternal morbidity (SMM) in Black and White patients with preeclampsia diagnosis versus signs/symptoms. STUDY DESIGN: This was a retrospective cohort study analyzing data from the IBM® Explorys Database between 7/31/2012-12/31/2020. Demographic, clinical, and laboratory data were extracted. Healthcare utilization and SMM were analyzed during the antepartum period (20 weeks of gestation until delivery) among Black and White patients with signs/symptoms of preeclampsia, with a diagnosis of preeclampsia, or neither (control). MAIN OUTCOME MEASURES: Healthcare utilization and SMM in those with a preeclampsia diagnosis or signs/symptoms of preeclampsia only were compared with a control group (White patients with no preeclampsia diagnosis or signs/symptoms). RESULTS: Data from 38,190 Black and 248,568 White patients were analyzed. Patients with preeclampsia diagnosis or signs/symptoms were more likely to visit the emergency room compared to those without diagnosis or signs/symptoms. Black patients with signs/symptoms of preeclampsia had the highest elevated risk (odds ratio [OR] = 3.4), followed by Black patients with a preeclampsia diagnosis (OR = 3.2), White patients with signs/symptoms (OR = 2.2), and White patients with a preeclampsia diagnosis (OR = 1.8). More Black patients experienced SMM (SMM rate 6.1% [Black with preeclampsia diagnosis] and 2.6% [Black with signs/symptoms]) than White patients (5.0% [White with preeclampsia diagnosis] and 2.0% [White with signs/symptoms]). SMM rates were higher for Black preeclampsia patients with severe features than for White preeclampsia patients with severe features (8.9% vs 7.3%). CONCLUSIONS: Compared with White patients, Black patients had higher rates of antepartum emergency care and antepartum SMM.

Oxytocin receptors in the primate ovary: molecular identity and link to apoptosis in human granulosa cells.

BACKGROUND: Oxytocin (OT) is produced by granulosa cells (GCs) of pre-ovulatory ovarian follicles and the corpus luteum (CL) in some mammalian species. Actions of OT in the ovary have been linked to luteinization, steroidogenesis and luteolysis. Human IVF-derived (h)GCs possess a functional OT receptor (OTR), linked to elevation of intracellular Ca(2+), but molecular identity of the receptor for OT in human granulosa cells (hGCs) and down-stream consequences are not known. METHODS AND RESULTS: RT-PCR, sequencing and immunocytochemistry identified the genuine OTR in hGCs. OT (10 nM-10 microM) induced elevations of intracellular Ca(2+) levels (Fluo-4 measurements), which were blocked by tocinoic acid (TA; 50 microM, a selective OTR-antagonist). Down-stream effects of OTR-activation include a concentration dependent decrease in cell viability/metabolism, manifested by reduced ATP-levels, increased caspase3/7-activity (P < 0.05) and electron microscopical signs of cellular regression. TA blocked all of these changes. Immunoreactive OTR was found in the CL and GCs of large and, surprisingly, also small pre-antral follicles of the human ovary. Immunoreactive OTR in the rhesus monkey ovary was detected in primordial and growing primary follicles in the infantile ovary and in follicles at all stages of development in the adult ovary, as well as the CL: these results were corroborated by RT-PCR analysis of GCs excised by laser capture microdissection. CONCLUSIONS: Our study identifies genuine OTRs in human and rhesus monkey GCs. Activation by high levels of OT leads to cellular regression in hGCs. As GCs of small follicles also express OTRs, OT may have as yet unknown functions in follicular development.

Ovarian acetylcholine and ovarian KCNQ channels: insights into cellular regulatory systems of steroidogenic granulosa cells.

Acetylcholine (ACh) may be an ovarian signaling molecule, since ACh is produced by non-neuronal granulosa cells (GCs) derived from the antral follicle, and likely also by their in vivo counterparts in the growing follicle. Furthermore, muscarinic ACh receptors (MR) are present in GC membranes and in cultured human GCs a number of MR-mediated actions have been described, including regulation of proliferation and gap junctional communication. Importantly, muscarinic stimulation elevates intracellular calcium levels, thereby opening a calcium-activated potassium channel (BK(Ca)) and causing membrane hyperpolarization. In the course of electrophysiological experiments with human GCs we also observed a reversible inhibitory action of an ACh analogue (carbachol) on an outward potassium current. This current is reminiscent of a so-called M-current described in neuronal systems, of which muscarinic regulation is well-known. Indeed, the current is sensitive to the specific KCNQ blocker XE991 and a possible underlying channel, KCNQ1 (K(v)7.1/K(v)LQT1) was detected by RT-PCR in GCs and by immunohistochemistry in large ovarian follicles. Pharmacological inhibition of the channel by XE991 blocked gonadotropin-stimulated steroid production and increased cell proliferation, i.e. fundamental processes of GCs in the ovary. Assuming a similar effect of ACh in vivo, this channel may be a pivotal regulator of physiological GC function linked to actions of the novel intraovarian signaling molecule ACh.

Non-neuronal acetylcholine and choline acetyltransferase in oviductal epithelial cells of cyclic and pregnant pigs.

Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct.

186Re radioimmunotherapy of small cell lung carcinoma xenografts in nude mice.

A 186Re-labeled monoclonal antibody (MAb), NR-LU-10, was used for the radioimmunotherapy of a subcutaneous human small cell lung carcinoma xenograft, SHT-1, in nude mice. Biodistribution with specific and irrelevant labeled MAb demonstrated peak tumor uptake of 8% and 3% of the injected dose/g at 2 days, respectively. Dosimetry analysis predicted tumor:whole-body radiation-absorbed dose ratios of 2.43:1 for NR-LU-10 and 0.62:1 for irrelevant MAb. Single-dose toxicity screening estimated a 50% lethal dose within 30 days of 600 microCi (880 cGy of whole-body radiation). As anticipated, a multiple-dose regimen of 490 microCi in four doses over 10 days (720 cGy of whole-body radiation, eight of eight surviving greater than 30 days) was less toxic than a single bolus dose of 430 microCi (644 cGy of whole-body radiation), six of eight surviving greater than 30 days). A multidose radioimmunotherapy regimen was initiated in nude mice bearing 66-mm3 tumors (total dose, 500 to 600 microCi). Complete remissions (greater than 140 days) were achieved in three of 16 mice, and the remainder showed a mean tumor growth delay of 53 days. Matched doses with irrelevant MAb produced one remission, one treatment-related death, and a mean growth delay of only 20 days in six of eight mice. Thus, in this nonoptimal radioimmunotherapy model, significant antitumor responses were observed using a mildly toxic multiple dosing regimen.

L-DOPA in the hu man ovarian follicular fluid acts as an antioxidant factor on granulosa cells.

BACKGROUND: A previous study showed that dopamine (DA), which is contained in follicular fluid (FF) from IVF patients, strongly increased the production of reactive oxygen species (ROS) by cultured human granulosa cells (GCs). ROS, including H2O2, are assumed to play roles in ovarian physiology and pathology. Ovarian DA could be derived from the circulation, ovarian innervation and/or unknown ovarian sources. L-DOPA is the direct precursor of DA in its synthetic pathway. It was not yet described in FF. We examined L-DOPA levels in FF from IVF patients. As it may exert anti-oxidative and ROS-scavenging functions, we studied whether it exerts such actions in human GCs and whether DOPA-decarboxylase (DDC), the enzyme converting L-DOPA to DA, is expressed in the human ovary. RESULTS: ELISA measurements revealed that human IVF-derived FF contains L-DOPA. In cultured human GCs automated confluence analyses showed that L-DOPA enhanced their survival. This is in contrast to the actions of DA, which reduced cell survival. A dose-dependent mode of action of L-DOPA was identified using a fluorescent ROS indicator. The results showed that it antagonized intracellular ROS accumulation induced by exogenous H2O2. DDC was absent in follicular GCs, but immunohistochemistry identified it in theca cells (TCs) of large follicles in the human ovary. Laser micro-dissection followed by RT-PCR corroborated the expression. DDC was also identified in the steroidogenic cells of the corpus luteum. CONCLUSIONS: L-DOPA in FF is an antioxidant factor and exerts positive influences on GCs. Ovarian DA is derived from L-DOPA and has opposite actions. Exogenous L-DOPA is a standard therapy for Parkinson's disease, and the results raise the possibility that it may be able to exert positive actions as an antioxidant in ovarian conditions, as well.

Photodynamic membrane damage at the level of single ion channels.

Illumination of cellular membranes by visible light in the presence of appropriate photosensitizers is known to inactivate specific ionic pathways and to increase the unspecific leak conductance of the membranes. While previous studies have concentrated on the macroscopic ionic currents, the present study separates the two phenomena at the microscopic level. Using opossum kidney (OK) cells as epithelial model system and photofrin II as sensitizer, the patch-clamp technique in inside-out configuration has been applied to show the inactivation of single ion channels immediately after start of illumination and the subsequent strong increase of the leak conductance. Inactivation is shown for two kinds of channels: the large-conductance Ca2+-dependent K+ channel (maxi-K(Ca)) and the stretch-activated nonselective cation channel (SA-cat).

Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Infections with Acinetobacter calcoaceticus (Herellea vaginicola): clinical and laboratory studies.

In a retrospective review of 53 patients, 58 episodes of infection due to Acinetobacter calcoaceticus var. anitratus (Herellea vaginicola) were studied. Although the organism is widely distributed in nature, it is of relatively low virulence since colonization is more frequently noted than infection and since most infections occur in patients subjected to the epidemiologic pressures common to nosocomial, gram-negative bacillary infection: prior antibiotic therapy; instrumentation and manipulation (e.g., endotracheal intubation, urinary bladder catheterization, arterial and venous cannulation); surgery; hospitalization, especially with residence in an intensive care unit; severe underlying disease, either systemic (e.g., chronic obstructive pulmonary disease, malignancy) or localized to the infected area (e.g., prior bacterial or aspirational pneumonia, trauma). Pneumonia was the most common infection due to A. calcoaceticus, and occurred only in patients with a tracheostomy or endotracheal tube in place. In over half the 25 patients, more than one lobe was involved and bronchopneumonia was the usual roentgenographic appearance. Cavitation (2 patients) and empyema formation (3 patients) were uncommon. The severity of acinetobacter pneumonia is reflected in the high mortality rate (44% overall, with a 36% mortality rate due primarily to infection). Tracheobronchitis due to A. calcoaceticus was less severe than pneumonia since no patients died primarily as a result of the infection. Urinary tract infections occurred in five patients, none of whom were ill and none of whom died. Urinary bladder catheterization was thought to be responsible for infection in three patients, and in at least four of the five patients infection was restricted to the lower tract. Wound infections were noted in six patients who had undergone surgery and were related to the presence of foreign bodies in the operative site in five of the patients. Surgical debridement and/or drainage of the infected area was the primary therapeutic measure employed in most cases. Only one patient died and this was a result of noninfectious causes. Skin infection due to A. calcoaceticus was seen in two patients, one of whom exhibited fulminant, fatal cellulitis and septicemia in the setting of pancytopenia. All nine patients with acinetobacter septicemia had received antecedent antibiotic therapy, and in all cases intravenous catheters were in place at the time bacteremia occurred. Clinically, seven of the nine patients were in shock. The mortality rate was 44% overall, with a 22% mortality rate due to infection. Although septicemia was thought to be "line-related" in five of the nine patients, serious post-bacteremic complications developed in three patients: prosthetic valve endocarditis, suppurative thrombophlebitis and subhepatic abscess.

Torulopsis glabrata fungemia--a clinical pathological study.

The clinical findings, pathologic features, and outcome were investigated in 46 patients in whom Torulopsis glabrata was isolated in 131 specimens of blood. Nineteen of the patients had only a single positive blood culture and no evidence of systemic yeast infection, while 27 patients had a clinically significant fungemia based upon the occurrence of 2 or more positive blood cultures, or the combination of a positive blood culture and isolation of the organism from a closed body cavity or demonstration of the yeast in tissue sections. The predisposing factors to the development of fungemia included the presence of intravenous lines, indwelling Foley catheters, antibiotics and surgery, especially when the gastrointestinal tract was involved. Only 22% of patients received either steroids or cytostatic agents. Possible portals of entry were suggested by the prior isolation of the organism from urine, sputum, wounds, and central venous catheter tips in most of the patients. Twelve of 27 patients with clinically significant fungemia were treated. The initial mode of therapy in nine patients was removal of intravenous lines because of the clinical suspicion of catheter related sepsis. Seven of the patients improved rapidly and one more after amphotericin B was subsequently administered. Amphotericin B was the initial therapy in three cases. One patient was cured while another died of an unrelated infection. Five patients were not treated although the isolation of T. glabrata had been reported; the fact that the presence of the organism was felt to be unimportant was considered to be a factor in the delay of treatment. In the remaining 10 patients the organism was isolated only after the patient had died. Division of the patients into four groups based upon whether the individuals survived, died of unrelated disease, died with potentially lethal infection, or died with T. glabrata infection significantly contributing to death, revealed a spectrum of disease, certain signs of which appeared to be of predictive value as prognostic indices of survival and severity of the infection. Seven patients with transient fungemia experienced an acute episode of high spiking fever (greater than 102.5 degrees F), rigors and/or hypotension, six of whom improved after the intravenous catheter was removed, suggesting a catheter-related sepsis. In contrast, persistent low grade fever (less than 102.5 degrees F) characterized eight of the nine patients in whom T. glabrata infection was considered either potentially lethal, or contributing significantly to death. A deteriorating clinical course with organ failure was also associated with this latter category of patients. Catheter-induced specticemia was considered in only two patients in this category. The autopsy and clinical findings in this investigation as well as reported experimental studies suggest that T. glabrata is an organism of low virulence. The patients' underlying disease (e.g., neoplasia) and coexisting bacterial infection are the most important factors responsible for death.

Comparison of Freund's and Ribi adjuvants for inducing antibodies to the synthetic antigen (TG)-AL in rabbits.

Antibody responses and health parameters were compared in rabbits immunized with a synthetic polypeptide antigen, [L-Tyr,L-Glu,DL-Ala]-poly-L-lysine ((TG)-AL), in Freund's (FA) or Ribi (RA) adjuvants. Rabbits, 12 weeks old, of both sexes, were inoculated with 0.5 ml divided between two intramuscular (i.m.) sites. Eight received FA and antigen (50 micrograms); eight RA and antigen, eight PBS and antigen; four FA and PBS; four RA and PBS, and four PBS. Identical booster inoculations were made 21 days later, except that incomplete FA was substituted for complete FA. Rabbits were monitored until euthanasia and necropsy 7 weeks after the primary inoculation. Sera, obtained weekly, were analyzed for immunoglobulins using an enzyme immunoassay. Only rabbits given antigen with adjuvant produced high titered antibodies. Mean optical density values for immunoglobulin (Ig)M were greater the week after the booster in the group given FA. IgG values were similar for both adjuvant/antigen groups the week after the booster, but thereafter decreased in rabbits given RA. Antisera from rabbits given antigen with FA had greater avidity for the antigen than that from rabbits given antigen with RA, however, the difference was not significant (p greater than 0.05). Rabbits inoculated with FA and antigen had high serum creatinine kinase levels the day after inoculation, showed evidence of discomfort, and extensive granulomatous inflammation at the inoculation sites. Lesions were minimal to mild in rabbits given antigen with RA and PBS with either adjuvant. While RA did not result in adverse side effects, the IgG response to (TG)-AL with RA was transient compared to FA.

Resistance to gentamicin, tobramycin and amikacin among clinical isolates of bacteria.

Susceptibility to the administration of gentamicin, tobramycin and amikacin was determined for all isolates of aerobic and facultative gram-negative bacilli submitted for testing to the clinical bacteriology laboratory of the Massachusetts General Hospital between July 1, 1974, and June 30, 1976. In this 24-month period more than 46,000 isolates of bacteria were tested by the single-disc diffusion (Bauer-Kirby) method. Resistance to one or more of the aforementioned aminoglycosidic aminocyclitol antibiotics was found among 4,114 stains. Correlation with quantitative susceptibility test methods revealed that disc-diffusion methods using 10 microng discs accurately predicted resistance to gentamicin and tobramycin, but overestimated the prevalence of resistance to amikacin by 20 to 60%. Most of the gentamicin-resistant Enterobacteriaceae in this study were also cross-resistant to tobramycin but were susceptible to amikacin. Many gentamicin-resistant strains of Ps. aeruginosa were susceptible to both tobramycin and amikacin. Resistance to amikacin tended to be of relatively low magnitude (most had minimal inhibitory concentrations (MIC's) between 31 and 125 microng/ml), but organisms which were resistant to the administration of amikacin were usually resistant to the other two aminoglycosidic antibiotics as well.

Effect of microencapsulation on oxygen distribution in islets organs.

In islet transplantation, limitation of oxygen supply may restrict graft function, particularly when encapsulated tissue is used. Therefore, oxygen tensions (PO2) in isolated islet organs (Brockmann bodies) of Osphronemus gorami were measured. In a thermostatically (37 degrees C) controlled measuring chamber, PO2 values were recorded at subsequent microelectrode positions on a radial track toward the center of the organ. In 2 independent groups, we studied the effect of fluid convection (n = 12) and microencapsulation (n = 12). In both groups, sigmoidal PO2 profiles were found, which permit differentiation in an oxygen-depleted zone surrounding the surface, a steep decline inside the tissue corresponding to the oxygen-consuming rim, and a plateau in the center without oxygen consumption which reflects necrosis. The PO2 values decreased (P < 0.001) when convection was stopped. Compared with starting values, PO2 levels at the surface were 61 +/- 3% with and 41 +/- 4% without convection. Surface values for encapsulated tissue were 44 +/- 5% compared with 64 +/- 4% in nonencapsulated tissue. In the tissue, center oxygen dropped to 27 +/- 5% with convection and to 6 +/- 3% without, and to 11 +/- 3% for encapsulated tissue compared with 22 +/- 4% for nonencapsulated tissue. The thickness of the outer oxygen-depleted zone was 81 +/- 16 microns with and 196 +/- 57 microns without convection (P < 0.001), and 188 +/- 16 microns for encapsulated and 94 +/- 14 microns for nonencapsulated tissue (P < 0.001). The oxygen-consuming rim was 295 +/- 22 microns with and 235 +/- 36 microns without convection (NS), and 216 +/- 15 microns for encapsulated and 315 +/- 24 microns for nonencapsulated tissue (P < 0.01). These results illustrate the special distribution of oxygen in isolated islet tissue and indicate that barium alginate encapsulation may worsen oxygenation mainly by expanding the "unstirred water layer" surrounding the tissue.

Computerization in microbiology.

Computerization of the clinical microbiology laboratory is finally coming of age. Operations and functions easily adapted from other clinical laboratories have already been implemented in the microbiology laboratory, e.g., fiscal, clerical, and other administrative housekeeping chores. Similarly, storage, retrieval, and analysis of banks of data easily collected and filed have also been successfully accomplished in microbiology for several years. The more challenging problems of computerization still deserve and require the attention of microbiologists. These problems lie in the smooth sequential formulation and transmission of clinical microbiological test results from the laboratory to clinical records; the manipulation of relevant data by the computer prospectively for the detection and prediction of nosocomial infections or miniepidemics; and the development of programed lessons and examinations for computerized instruction, retraining, and examination of technologists and other individuals interested in microbiology.

Effects of penicillin and lysozyme on the immunofluorescent and precipitin reactivity of group D streptococci.

Damage to the cell wall by growth in the presence of penicillin or by treatment with lysozyme enhanced the immunofluorescent (fluorescent antibody, FA) reactivity to group D streptococci. The optimum concentration and time of treatment with lysozyme varied inversely with the initial FA reactivity of the strain. Speciation of the organisms by a series of biochemical and physiologic tests suggested that the differences in initial FA reactivity were species-related. Thus, S. faecalis strains were the most FA-reactive and most sensitive to lysozyme. S. faecium strains were less FA-reactive and lysozyme-sensitive. S. bovis strains proved to be least FA-sensitive and were most resistant to lysozyme. Treatment with lysozyme was also effective in preparing extracts of group D antigen from all three species for Lancefield grouping by the precipitin test. The lysozyme extracts, moreover, produced much stronger reactions than those made from comparable volumes of cells by the methods of Lancefield or of Rantz and Randall.

Distribution and incidence of viridans streptococcal species in routine clinical specimens.

Five hundred consecutive isolates of viridans streptococci were identified to the species level in an effort to determine their distribution and incidence in routine clinical specimens. Viridans streptococci accounted for significant percentages of streptococcal isolates from urine, wounds, body fluids, and blood. The most commonly isolated strains belonged to the Streptococcus milleri, Streptococcus mitis, Streptococcus sanguis I, and Streptococcus sanguis II species. Patient charts were reviewed in order to investigate the possible role as a urinary pathogen of strains belonging to a subgroup of S. milleri. Although these strains frequently are isolated from urine, they appear to play no pathogenic role in urinary tract infections.

Intra-abdominal Citrobacter infections: association with biliary or upper gastrointestinal source.

The clinical records of 46 patients with intra-abdominal and/or bacteremic infections caused by Citrobacter organisms were reviewed to determine whether the isolation of Citrobacter organisms from these sites could be used to determine its portal of entry. The isolation of Citrobacter organisms from intra-abdominal or pleural fluid cultures was associated with a biliary tract or upper gastrointestinal source in 36 of 41 patients. The biliary tract or small bowel was likewise the portal of entry for 8 to 13 patients with Citrobacter septicemia. Thus, the isolation of these organisms from the blood, pleural space, or abdominal cavity strongly suggests that the upper gastrointestinal (or biliary) tract is the site of significant pathologic disease. Of the antibiotics tested, moxalactam, cefotaxime, and amikacin were most effective against Citrobacter.