Application of disaccharides alone and in combination,for the improvement of stability and particle properties of spray-freeze dried IgG

Purpose: Spray-freeze drying (SFD) is a recently applied method to develop pharmaceutical powders. This study aimed to analyze the competence of Trehalose, Mannitol, Lactose, and Sorbitol instability and aerosolization of Immunoglobulin G (IgG) via SFD.

Methods: Induced soluble aggregates were quantified at 0 and 3?months, and 45?°C using size-exclusion chromatography. Conformation and thermogravimetric assessments were done by Fourier transform infrared spectroscopy and differential scanning calorimetry. Laser light scattering was performed to determine the particle sizes. Aerodynamic features were characterized by twin stage impinger and scanning electron microscopy.

Results: Although sugars/polyols preferably stabilized IgG following the process, storage stabilization was achieved in Trehalose, Trehalose-Lactose, Lactose, and Trehalose-Mannitol-based powders with soluble aggregates <5%. The conformation of antibody was preserved with β sheet content from 66.28% to 76.37%. Particle sizes ranged from 5.23 to 8.12?µm. Mannitol exhibited the best aerodynamic behavior, fine particle fraction (FPF: 70%) but high degree of protein aggregation during storage.

Conclusions: SFD could favorably stabilize antibody using Trehalose and its combination with Lactose and Mannitol, and also, Lactose alone. Sorbitol disturbed IgG powder recovery. Incorporation of other types of excipient is required for efficient respiratory delivery of IgG molecules.     

Mutational analysis of enhancer domains responsive to trans-activation by the X gene of human hepatitis B virus

Summary The X gene product of human hepatitis B virus (HBV) trans-activates the HBV enhancer. In order to identify domains responsive to trans-activation by the X gene, we introduced a series of mutations into the HBV enhancer and assayed the enhancer activities in the presence of the X gene product. Our results suggest that the EP domain of the enhancer is essential for trans-activation by the X gene.     

Inhibition of inducible nitric oxide synthase enhances anti-tumour immune responses in rats immunized with IFN-gamma-secreting glioma cells

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL), and non-selective, N-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l-NIL and l-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma, and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with l-NIL, but not l-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, l-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.     

Infradiaphragmatic versus supradiaphragmatic Hodgkin lymphoma: a retrospective review of 1,114 patients

Infradiaphragmatic Hodgkin lymphoma (IDH) accounts for 4-13% of cases of stage I-II Hodgkin lymphoma (HD). It has been associated with distinct pre-treatment characteristics and outcomes when compared with supradiaphragmatic HD (SDH). The comparison of IDH vs SDH can only be made in early and intermediate stages (I-II), such a comparison is not possible for advanced stages (III-IV). This study retrospectively compared two groups of 1013 patients with stage I-II SDH and 101 patients with IDH (10%). These two sub-groups of patients were treated in 1988-1993 in 2 prospective randomized clinical trials in Germany for early and intermediate stages of Hodgkin lymphoma. IDH-patients were older (median 39 vs 31 years; p < 0.001), predominantly male (73% vs 52%; p < 0.001) and more often had involvement of 3 lymph node areas (LNA) (80% vs 55%; p < 0.001). Histology in IDH was more likely to be mixed cellularity (46.5% vs 23.6%, p < 0.001) or lymphocyte predominant (20 vs 10%, p = 0.003) and less likely nodular sclerosis (25% vs 63%, p < 0.001). In early-stage unfavorable disease, IDH was associated with a higher treatment failure rate (unadjusted hazard ratio 2, 95% CI, 1.3-3.4; p = 0.003). After controlling for age, sex, stage, histology, B-symptoms and involvement of 3 LNA, the adjusted hazard ratio was 1.25 (95% CI, 0.65-2.4; p = 0.51) so that IDH was no longer associated with a statistically significant treatment failure rate. Poorer outcomes with IDH as compared to SDH are attributable to its association with known adverse prognostic risk factors, but IDH, in itself, is not an independent adverse prognostic factor for treatment failure or survival.     

Childbearing among young Latino women in the United States.

We analyzed 1979 and 1982 data from the Youth Cohort of the National Longitudinal Surveys (NLS) of Labor Market Experience to compare rates of early childbearing among White, Black, Mexican-origin and Puerto Rican women up to age 21. Latino young women fall in between the extremely low rate of the Whites and the extremely high rate of the Blacks. Mexican and Puerto Rican young women have similar proportions of premarital first births, but the marital first birth rate for young Mexicans is twice that of the Puerto Ricans. The bulk of Mexican first births, like births to Whites, occur within marriage, while Puerto Rican first births are similar to those of Blacks, the majority being out-of-wedlock. These racial/ethnic differences in premarital first birth rates do not change greatly when socioeconomic status, and birthplace of respondents and respondents' parents are controlled.     

The outermost N‐terminal region of tapasin facilitates folding of major histocompatibility complex class I

Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC‐I biosynthesis. It binds MHC‐I molecules, integrates them into peptide‐loading complexes, and exerts quality control of the bound peptides; only when an “optimal peptide” is bound will the MHC‐I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn_1–87 (representing the 87 N‐terminal and ER‐luminal amino acids of the mature Tpn protein). Using a biochemical peptide–MHC‐I‐binding assay, recombinant Tpn_1–87 was found to specifically facilitate peptide‐dependent folding of HLA‐A*0201. Furthermore, we used Tpn_1–87 to generate a monoclonal antibody, αTpn_1–87/80, specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of αTpn_1–87/80 was located to Tpn_40–44, which maps to a surface‐exposed loop on the Tpn structure. Together, these results demonstrate that the N‐terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC‐I.     

The Callender modification of the Macintosh laryngoscope blade reduces the risk of tooth-blade contact in children

Background: Dental injury is well recognized as a potential complication of laryngoscopy and tracheal intubation. The flange of the Macintosh blade may contact teeth during laryngoscopy causing damage. The Callander modification of the Macintosh blade (Callander blade) with a shortened heel at the proximal end has been shown to increase the blade–tooth distance and reduce contact rates in adults. Aim: This study was designed to evaluate the effectiveness of the Callander blade on reducing dental contact in children. Methods: One hundred children, aged 4–14 years, scheduled for general anesthesia requiring endotracheal intubation were studied prospectively. The children were preoperatively evaluated for Mallampati score and dental defects, looseness, and avulsions. Following induction of anesthesia, laryngoscopy was performed twice on each child in succession, once with a standard Macintosh blade and once with a Callander blade of the same size in a random order. The blade–tooth distance and contact rate were compared between the two blades. Results: With the Callander blade, the blade–tooth distance was greater than with the Macintosh (1.78 ± 1.77 vs 0.28 ± 0.76 mm, P = 0.001). The frequency of direct contact was less with the Callander blade than it was with the Macintosh blade (33% vs 86%, respectively, P = 0.008). However, difficult laryngoscopy rate did not differ between the blades. Tracheal intubation was successful in all children and there was no dental injury. Conclusion: Our findings suggest that the Callander blade decreases the risk for dental injury and provides tracheal intubating conditions in children with normal airways similar to those obtained with a traditional Macintosh blade.