Novel case of combination antibiotic therapy for treatment of a complicated polymicrobial urinary tract infection with one organism harboring a metallo-ß-lactamase (MBL) in a pregnant patient.

Carbapenem resistance due to metallo-beta-lactamases (MBLs) is a global phenomenon and an important challenge for antibiotic therapy (Boyd et al., 2020 [1]). While previous reports have demonstrated both in vitro and in vivo synergy using the combination of ceftazidime-avibactam and aztreonam against Stenotrophomonas maltophilia, an MBL-harboring organism, this treatment strategy has not been reported during pregnancy (Mojic et al., 2017 [2], [3], Mojica et al., 2016 [4], Alexander et al., 2020 [5]). We describe a 33-year-old pregnant female with polymicrobial, bilateral pyelonephritis caused by Stenotrophomonas maltophilia and other gram-negative bacteria. The organisms were eradicated with the combination of ceftazidime-avibactam and aztreonam followed by successful delivery with no observed adverse effects in either mother or child post-partum.

In vivo evaluation of matrix pellets containing nanocrystalline ketoprofen.

The aim of this study was to evaluate the in-vivo behaviour of matrix pellets formulated with nanocrystalline ketoprofen after oral administration to dogs. No significant differences in AUC-values were seen between pellet formulations containing nanocrystalline or microcrystalline ketoprofen and a commercial ketoprofen formulation (reference: Rofenid 200 Long Acting). C(max) of the formulations containing nano- or microcrystalline ketoprofen was significantly higher compared to reference, whereas t(max) was significantly lower. The in-vivo burst release observed for the spray dried nanocrystalline ketoprofen matrix pellets was reduced following compression of the pellets in combination with placebo wax/starch pellets. These matrix tablets sustained the ketoprofen plasma concentrations during 5.6 and 5.4 h for formulations containing nano- and microcrystalline ketoprofen, respectively.

An oral controlled release matrix pellet formulation containing nanocrystalline ketoprofen.

A controlled release pellet formulation using a NanoCrystal colloidal dispersion of ketoprofen was developed. In order to be able to process the aqueous NanoCrystal colloidal dispersion into a hydrophobic solid dosage form a spray drying procedure was used. The in vitro dissolution profiles of wax based pellets loaded with nanocrystalline ketoprofen are compared with the profiles of wax based pellets loaded with microcrystalline ketoprofen and of a commercial sustained release ketoprofen formulation. Pellets were produced using a melt pelletisation technique. All pellet formulations were composed of a mixture of microcrystalline wax and starch derivatives. The starch derivatives used were waxy maltodextrin and drum dried corn starch. Varying the concentration of drum dried corn starch increased the release rate of ketoprofen but the ketoprofen recovery remained problematic. To increase the dissolution yield surfactants were utilised. The surfactants were either added during the production process of the NanoCrystal colloidal dispersion (sodium laurylsulphate) or during the pellet manufacturing process (Cremophor RH 40). Both methods resulted in a sustained but complete release of nanocrystalline ketoprofen from the matrix pellet formulations.

Neutrophil chemotaxis and superoxide production are induced by cross-linking FcgammaRII receptors.

Neutrophils express two types of receptor for the Fc region of IgG, FcgammaRII and FcgammaRIIIB. Via these receptors, neutrophils bind IgG complexes that contain more than one IgG molecule. This binding activates functional processes, such as the respiratory burst and chemotaxis. Neutrophils were treated with biotinylated anti-Fc receptor monoclonal antibodies and chemotaxis toward streptavidin, a cross-linking agent, was determined. Cross-linking FcgammaRII and not FcgammaRIIIB induced neutrophil chemotaxis. Superoxide production in response to immobilized anti-Fc receptor antibodies was also examined. Anti-FcgammaRII Fab bound to ELISA plates induced superoxide production, while anti-FcgammaRIIIB Fab did not. Pretreatment of neutrophils with anti-FcgammaRII Fab reduced superoxide generated by immobilized anti-FcgammaRII antibody. The data demonstrate that FcgammaRII and not FcgammaRIIIB are responsible for neutrophil chemotaxis and superoxide production upon Fc receptor activation.

Effect of allotype on activation of neutrophils by FcgammaRIIIB cross-linking.

Human neutrophils constitutively synthesize two receptors for the constant region of IgG, FcgammaRII, and FcgammaRIIIB. Fluo-3-loaded neutrophils were treated with biotinylated Fab fragments of anti-FcgammaR antibodies and cross-linked with streptavidin, and intracellular calcium ([Ca2+](i)) was monitored by flow cytometry. Polymerization of filamentous actin was quantitated by NBD-phallacidin using flow cytometry. Cross-linking of FcgammaRII by monoclonal antibody (mAb) IV.3 induces an increase in [Ca2+](i), superoxide generation, and the polymerization of actin. [Ca2+](i) responses from cross-linking of FcgammaRIIIB by mAb 3G8 varied from minimal to no release. To determine whether discrepancies in 3G8-induced [Ca2+](i) release were due to allotype variation, we selected five donors who were homozygous for the NA1 allotype of FcgammaRIIIB and five who were either heterozygous or homozygous for the NA2 allotype and compared their [Ca2+](i) response and actin polymerization induced by FcgammaRIIIB cross-linking. Cross-linking of FcgammaRIIIB by 3G8 produced minimal [Ca2+](i) release and polymerization of actin irrespective of donor allotype.

Myelinated dorsal root ganglion cultures activate both the alternative and classical pathways of complement.

We used rat myelinated dorsal root ganglion (MDRG) cultures to study antibody and complement-mediated mechanisms of peripheral demyelinating diseases. Heat inactivated serum from a patient (LT) with peripheral neuropathy and a monoclonal IgM reactive with myelin-associated glycoprotein (anti-MAG) and sulfated glucuronosyl glycolipids (anti-SGGL) was used as an antibody source. Incubation of whole human serum (WHS) or WHS and anti-SGGL with MDRGs resulted in reduction of classical and alternative pathway hemolytic activities and the development of abnormal myelin sheaths. Incubation of MDRG cultures with C2-deficient serum showed activation of the alternative complement pathway. Classical pathway hemolytic activity was reduced when Factor B-depleted serum was incubated with MDRG cultures. The rat MDRG culture system provides a good model system of a peripheral nerve and has therefore been used by several investigators to study antibody and complement-mediated demyelination associated with peripheral neuropathies. However, our studies indicate a high degree of complement activation and membrane disruption of cultures incubated with WHS.

Design and characterization of a surfactant-enriched tablet formulation for oral delivery of a poorly water-soluble immunosuppressive agent.

The feasibility of incorporating significant quantities of the anionic surfactant, sodium lauryl sulfate (SDS), into an immediate release tablet formulation of a poorly water-soluble immunosuppressive agent was investigated. Despite the extremely poor compressibility of SDS and poor chemical stability of the drug, a commercializable, direct-compression tablet formulation with satisfactory mechanical properties and acceptable chemical stability was achieved. Optimal in vitro release of the drug from the tablet formulation was achieved by establishing the minimum molar uptake ratio necessary to achieve complete micellar solubilization of the drug, after which formulation studies were conducted to determine the influence of formulation and process variables on the rate and extent of drug release. A model-independent analysis of dissolution results in a reduced volume (250 ml) of modified simulated gastric fluid demonstrated that the rate and extent of drug release was highly dependent on the mean particle size of the bulk drug, but independent of compression force above that required to achieve a compact of acceptable mechanical strength. Employing the Korsmeyer-Peppas model of Fickian and non-Fickian drug release, it was further shown that release of the drug from the dosage form was governed largely by surface erosion of the surfactant-enriched tablet matrix.

Effects of thrombospondin purified by heparin affinity or ion-exchange chromatography on the alternative complement pathway.

Thrombospondin (TSP), a platelet glycoprotein, was purified from platelet supernatants applied sequentially to Sepharose 4B and heparin-agarose affinity columns. This TSP inhibited alternative pathway activity in serum as assessed by lysis of rabbit erythrocytes and contained bound heparin, a substance which is known to inhibit the alternative pathway. TSP purified by anion exchange chromatography did not contain heparin and was not inhibitory. Chromatography of this TSP over a heparin-agarose affinity column resulted in TSP which contained heparin and inhibited the alternative pathway. Purification of TSP by the standard technique of heparin affinity chromatography results in preparations which are contaminated with heparin.

Changes in immunologic cell surface markers during cocaine withdrawal in pregnant women.

To evaluate the effects of acute cocaine withdrawal on the immune system of pregnant women, we analyzed changes in a panel of cell surface markers and plasma proteins that have immunological importance. The cell surface markers included complement receptors [CR1 (CD35), CR2 (CD21), CR3 (CD11b, CD18)], immunoglobulin Fc receptors [FcgammaRII (CD32), FcgammaRIII (CD16)], proteins important for lymphocyte function [CD3, CD4, CD8, CD19, CD25, CD45RA], and the framework antigen HLA-ABC. We also measured levels of the plasma proteins C3, C4, IgG, IgM, and IgA, along with the cytokine interleukin-2, soluble lymphocyte markers sCD25, sCD4, sCD8, IL-2, and soluble immune complexes. While no significant changes were seen in the levels of plasma proteins, changes paralleling the course of acute withdrawal were seen in complement receptors and immunoglobulin Fc receptors on leukocyte subpopulations. By contrast, proteins important for lymphocyte function were relatively unperturbed. There was an increase in receptor expression at the onset of withdrawal that peaked 3-5 days after last cocaine use, followed by a decrease in expression to initial (pre-withdrawal) levels. These changes in cell surface receptors may reflect altered immune function in the women who were withdrawing from cocaine.

Rheumatic diseases and inherited complement deficiencies.

Deficiencies of individual complement proteins may be accompanied by SLE or related syndromes. Deficiencies of the classic activation pathway are often involved. In cases of C4 and C2 deficiency, there is evidence that this association occurs more frequently than would be expected by chance. The clinical picture differs from classic SLE. There is an increased frequency of skin involvement, a decreased frequency of renal disease, low or absent levels of antibody to native DNA, and increased levels of anti-Ro (SS-A). The mechanism for the association probably involves the effects of C3 and C4 on the precipitation of immune complex solubility, or on their processing through cell surface c4b/c3b receptors on phagocytes. Disseminated or recurrent Neisseria infections are common in patients lacking the constituents of the terminal MAC that are important in killing these organisms.

B lymphocytic clonal expansion in rheumatoid arthritis.

OBJECTIVE: To learn whether rheumatoid factor (RF), HLA-DR4, or current therapy influences clonal expansion of B lymphocytes (B cells) in persons with rheumatoid arthritis (RA). METHODS: We measured clonal expansion by analysis of cell surface staining for immunoglobulin light chains. Double staining methods detected a B cell marker (CD19) plus either kappa or lambda on peripheral blood lymphocytes from subjects with RA (n = 26) and controls (n = 26). The difference between frequency histograms of surface kappa and lambda staining was determined by the Kolmogorov-Smirnov statistic D that represents the fraction of clonally expanded B cells. RESULTS: The mean D value in RA was over 50% higher than in the controls [0.225 +/- SD 0.155 versus 0.144 +/- 0.025 (p < 0.0001)]. Ten subjects with RA had values exceeding +2 SD for controls (p = 0.0007). Mean D correlated with RF titer (Spearman's rank correlation coefficient rSp + 0.53, p = 0.006). All 10 high D values were found in the RA subgroups with positive serum tests for RF and with the HLA-DR4 positive genotype. The channel of maximal difference between kappa and lambda staining was higher in the RA group than in controls, showing that clonal expansion was most marked among brightly staining cells. Patients with RA currently receiving low dose methotrexate (MTX) tended to have higher D values than those not receiving MTX (mean 0.29 versus 0.18, respectively, p < 0.025). The RA group currently receiving MTX had a higher frequency of abnormal D values (7 of 11 versus 3 of 15 not currently receiving MTX, p = 0.03). This probably reflects preferential use of MTX for severely affected individuals. Confirmatory studies to detect clonal immunoglobulin gene rearrangements were attempted in selected individuals with high D values, but none was demonstrated in total leukocytic or B cell enriched fractions. CONCLUSION: Findings consistent with B cell clonal expansion occur in about 40% of persons with RA, particularly in the subgroups with positive serum tests for RF and with the HLA-DR4 genotype. However, the clonal expansion level must be below the sensitivity of confirmatory methods.

Covalent linkage of C3 to properdin during complement activation.

Activation of the alternative complement pathway of serum produces complexes of properdin (P) and C3 as measured in a double antibody enzyme-linked immunosorbent assay. When purified from serum, these complexes decrease factor B hemolytic activity in serum and do not restore the alternative pathway hemolytic activity of serum deficient in P. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of activated serum containing biotinylated P, followed by blotting to nitrocellulose and development with streptavidin-alkaline phosphatase revealed a band at 53 kDa for monomeric P and an additional band at 160 kDa. P samples eluted from zymosan and purified from activated serum revealed a band at 116 kDa for C3 alpha and 74 kDa for C3 beta, and an additional band at 160 kDa when analyzed by SDS-PAGE, Western blotting and development with antibody to C3. The appearance of a 160 kDa band containing P and C3 indicates that these proteins are contained in a complex formed during activation of the alternative pathway. Activation of a purified reagent mixture containing factors B, D, and H, and 125I-labeled P or 125I-labeled C3, followed by SDS-PAGE and autoradiography confirmed the presence of a 160-kDa band which disappeared following hydroxylamine treatment of the sample. These data are consistent with a covalent linkage of C3 to P via the C3 alpha chain, producing the 160-kDa complex.

Successful cyclophosphamide treatment of cryoglobulinemic membranoproliferative glomerulonephritis associated with hepatitis C virus infection.

A 54-year-old man with cryoglobulinemia and chronic hepatitis C infection presented with progressive renal insufficiency caused by membranoproliferative glomerulonephritis. Because of a steady decline in renal function, cyclophosphamide therapy was instituted. Within 1 month of starting therapy, his cryoglobulins disappeared, and in 3 months, his creatinine clearance had improved from 56 mL/min to 89 mL/min. At no point in his course was there clinical evidence of liver disease. After 1 year, cyclophosphamide was successfully stopped. Fourteen months later, his creatinine clearance is 105 mL/min. These results suggest that cyclophosphamide may be useful therapy for patients with cryoglobulinemic membranoproliferative glomerulonephritis and hepatitis C virus infection who have progressive renal insufficiency.

Transient lupus anticoagulant associated with prothrombin deficiency: unusual cause of bleeding in a 5-year-old girl.

PURPOSE: We present the association of a lupus anticoagulant with hypoprothrombinemia in a 5-year-old girl, who presented with ecchymoses and a hematoma. This coagulopathy should be included in the differential of bleeding in the previously healthy children. PATIENTS AND METHODS: Coagulation and immunology laboratory evaluation was performed at the time of presentation with bleeding and 2 months later, after complete clinical recovery. RESULTS: A 5-year-old girl presented with ecchymoses and a hematoma after after an upper respiratory illness. Laboratory evaluation showed prolongation of both the prothrombin time (PT) and activated partial thromboplastin time (aPTT) due to the presence of a strong lupus anticoagulant associated with a decreased level of prothrombin (15 U/dl). Hypocomplementemia was also detected. Bruising resolved spontaneously, and the PT and aPTT gradually normalized. Reevaluation 2 months later showed that the lupus anticoagulant had disappeared and the prothrombin deficiency was markedly improved. CONCLUSIONS: This case demonstrates that transient lupus anticoagulants must be included in the differential for bleeding in young children. Also, in children with lupus anticoagulants, neither the association of hypoprothrombinemia nor the presence of evidence of activation of the immune system appears to predict whether a patient will have or develop systemic lupus erythematosus.

Restricted expression of a zinc finger protein in male germ cells.

Zfp-37 is a zinc finger protein gene expressed in male germ cells. The cDNA detected two transcripts on Northern blots of testis RNA, with expression first detected at around day 19. To establish the pattern of expression of the protein we have raised antibodies to ZFP-37 and used them on thin sections of testis and on Western blots. On Western blots the antibody detected two proteins exclusively in testis extracts, confirming the previous mRNA expression data. A time-course study revealed that the larger of the two proteins appears at about day 22 but the smaller one is not detected until day 34. Analysis of the expression of these two proteins in purified germ cell preparations revealed that the smaller protein is only detectable in the elongating spermatids or residual bodies. Data from thin sections showed that most, but not all, of the protein recognized by the antibody is in the nucleus, a result further confirmed by Western blotting. These results are discussed in the light of the possible role of this protein in regulating spermatogenesis.

High-performance liquid chromatographic method for the simultaneous determination of low-molecular-mass oligomers of polyethylene glycol in aqueous skin extracts.

The isocratic, reversed-phase, high-performance liquid chromatographic (HPLC) method presented provides a simple and rapid analytical technique for the simultaneous determination of low-molecular-mass oligomers of polyethylene glycol (PEG) in aqueous polymer samples containing polar contaminants of biologic origin. In the present case, individual molecular mass species were quantitated in aqueous skin extracts arising from the investigation of PEG transport through mammalian skin. PEGs were isolated and purified by solid-phase extraction using large-pore kieselguhr (Extrelut QE) cartridges, which selectively retained polar contaminants from excised skin specimens. Sample recoveries were found to be dependent upon molecular mass, ranging from 18.28% to 86.10%, but were highly reproducible. The mean inter-sample error (n > or = 5) for the extraction of twenty-six molecular mass species of PEG was less than 3%. Satisfactory resolution of primary molecular mass components was accomplished using a base-deactivated, C8 column (Supelcosil LC-8-DB) and a mobile phase consisting of methanol and water. Corresponding run times for the complete separation of individual oligomers ranged from 8 to 30 min, while the limit of detection for a particular molecular mass species was approximately 5 micrograms/ml. The method was further employed to determine the weight-average (Mw) and number-average (Mn) molecular mass distributions and polydispersity for three commercial PEG blends, as well as to characterize the methylene chloride/water distribution coefficients for twenty-two molecular mass species ranging from 282 to 1206 Da.

The mouse t-complex gene, Tcp-11, is under translational control.

The mouse t-complex is known to harbour genes which affect male fertility. Tcp-11 is a t-complex gene which is only expressed in male germ cells and from its position is a candidate for a distorter, one of the two types of genetic element involved in transmission ratio distortion. Antibodies raised to TCP-11 protein made in E. Coli were used on thin sections of testis and shown to recognise late spermatids. On Western blots the antibodies bound to a 68-kD protein present in protein extracts from testis. No specific signal could be detected using the antibody on protein extracts from other mouse tissues. Following gentle lysis of the germ cells and fractionation on sucrose gradients, all the material recognised by the anti-Tcp-11 antibody was found to be soluble and unassociated with any membrane fraction or organelle. A comparison of the time course of expression of the Tcp-11 mRNA and the TCP-11 protein revealed that expression of this gene is under translational control.