Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs.

A subunit protein vaccine candidate based on norovirus (NoV) virus-like particles (VLPs) and rotavirus (RV) VP6 protein against acute childhood gastroenteritis has been proposed recently. RV VP6 forms different oligomeric nanostructures, including tubes and spheres when expressed in vitro, which are highly immunogenic in different animal models. We have shown recently that recombinant VP6 nanotubes have an adjuvant effect on immunogenicity of NoV VLPs in mice. In this study, we investigated if the adjuvant effect is dependent upon a VP6 dose or different VP6 structural assemblies. In addition, local and systemic adjuvant effects as well as requirements for antigen co-delivery and co-localization were studied. The magnitude and functionality of NoV GII.4-specific antibodies and T cell responses were tested in mice immunized with GII.4 VLPs alone or different combinations of VLPs and VP6. A VP6 dose-dependent adjuvant effect on GII.4-specific antibody responses was observed. The adjuvant effect was found to be strictly dependent upon co-administration of NoV GII.4 VLPs and VP6 at the same anatomic site and at the same time. However, the adjuvant effect was not dependent on the types of oligomers used, as both nanotubes and nanospheres exerted adjuvant effect on GII.4-specific antibody generation and, for the first time, T cell immunity. These findings elucidate the mechanisms of VP6 adjuvant effect in vivo and support its use as an adjuvant in a combination NoV and RV vaccine.

You don't leave your baby--mother's experiences after a stillbirth.

When a baby has died during pregnancy, the first encounter between mother and child occurs when the baby is already dead. Despair, emptiness, and grief characterize the encounter, which is also a gradual farewell to the child and the planned future for the family. This study describes mothers' experiences of the farewell of their stillborn baby at discharge from hospital. Twenty-three mothers from different parts of Sweden, who suffered stillbirth, were interviewed. Semi-structured questions were used and the replies were analyzed using content analysis. The mothers describe the separation from the child when leaving hospital as unnatural and that the separation ruins the motherhood they felt during pregnancy. Five categories were identified: unnatural to leave the baby; going home empty-handed; access to the child; security and insecurity in the separation; to let go. The overarching theme that we recognized from these responses we have formulated as: You don't leave your baby. Leaving the baby at the hospital goes against the biological instinct to care for and protect the offspring. Routines for a dignified goodbye including designating a deputy guardian into whose arms the mother can place the baby can help to facilitate the separation. The possibility of leaving the baby in the arms of someone known to the parents should be an option for parents who choose to take farewell of the child at the hospital. The place and time for the farewell should be decided on by the parents, taking the baby home for a personal farewell could be an alternative.

Waiting in no-man's-land - mothers' experiences before the induction of labour after their baby has died in utero.

OBJECTIVE: Carrying death instead of life is beyond understanding and a huge psychological challenge for a pregnant mother. The aim of this study was to investigate the mothers' experiences of the time from the diagnosis of the death of their unborn baby until induction of labour. METHOD: In this qualitative study, in-depth interviews were conducted with 21 mothers whose babies had died prior to birth. The interviews were then analysed using content analysis. RESULTS: The overall theme that emerged from the mothers' experiences is understood as "waiting in no-man's-land", describing the feeling of being set aside from normality and put into an area which is unrecognized. Four categories were established: 'involuntary waiting' describes the sense of being left without information about what is to come; 'handling the unimaginable' concerns the confusing state of finding oneself in the worst-case scenario and yet having to deal with the birth; 'broken expectations' is about the loss not only of the baby but also of future family life; and 'courage to face life' describes the determination to go on and face reality. CONCLUSIONS: The mother's experiences during the time after the information of their baby's death in utero until the induction of labour can be understood as a sense of being in no-man's-land, waiting without knowing for what or for how long.

Losing contact with one's unborn baby--mothers' experiences prior to receiving news that their baby has died in utero.

BACKGROUND: A change in the pattern of movement of her unborn baby could be indicative that the baby might die. AIM: To study mothers' experiences during the time prior to receiving news that their baby has died. METHOD: Interviews with 26 mothers. RESULTS: Premonition that something had happened to their baby, a sense based on a lack of movements were experienced. Six categories describe the mother's insight that the baby's life was threatened: not feeling in touch with their baby; worry feeling something is wrong; not understanding the unbelievable; wanting information; and being certain that their baby had died. The overarching theme "There is something wrong" was formulated. CONCLUSION: The mother could not understand the unbelievable: that the baby had died in utero. IMPLICATIONS: Mother's should be cautioned to trust their insights and seek medical advice if they are concerned over the lack of movement from the unborn baby.

Determination of lamivudine, zidovudine, and nevirapine in capillary blood sampled on filter paper by LC.

A bioanalytical method for determination of lamivudine (3TC), zidovudine (AZT), and nevirapine (NVP) in 100 microL capillary blood applied onto sampling paper has been developed and validated. The antiretroviral drugs (ARV) were analyzed by reversed phase gradient liquid chromatography with UV detection. Separation was performed on a Zorbax SB C(8) (250 x 4.6 mm) column with a two-step gradient: (i) methanol-0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 15:85 v/v) and (ii) methanol-0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 50:50 v/v) with a flow rate of 1.0 mL/min. UV detection was performed at 260 nm. Total assay precisions were 6.3, 4.7, and 4.9% for 3TC at 0.34, 0.69, and 3.9 microg/mL, and 5.1, 5.5, and 3.2% for AZT at 0.40, 0.80, and 4.5 microg/mL. For NVP, total assay precisions were 5.2, 8.3, and 3.5% at 2.6, 4.5, and 8.8 microg/mL. Lower limit of quantifications (LLOQ) were 0.11 and 0.13 microg/mL for 3TC and AZT where the precisions were 2.0% for both the analytes. For NVP, LLOQ was 1.3 microg/mL where precision was 2.6%. Concentrations were determined for 10 h for two subjects receiving standard twice daily antiretroviral therapy containing 3TC, AZT, and NVP. Maximum 3TC concentrations were 2.5 and 2.8 microg/mL for subject 1 and 2, respectively. For AZT, maximum concentrations were 1.8 and 1.1 microg/mL while being 15 and 9.6 microg/mL for NVP. Pre-dose trough concentration of NVP was 11 microg/mL for subject 1 and 9.6 microg/mL for subject 2.

Straightforward and rapid determination of sulfadoxine and sulfamethoxazole in capillary blood on sampling paper with liquid chromatography and UV detection.

A method for the determination of sulfadoxine and sulfamethoxazole in capillary blood on sampling paper has been developed and validated. The method is straightforward with minimal sample preparation, and is suitable for rural settings. Separation of sulfadoxine, sulfamethoxazole and internal standard was performed using a Purospher STAR RP-18 endcapped LC column (150x4.6mm) with a mobile phase consisting of acetonitrile:sodium acetate buffer pH 5.2, I=0.1 (1:2, v/v). For sulfadoxine, the within-day precision was 5.3% at 15micromol/l and 3.7% at 600micromol/l, while for sulfamethoxazole it was 5.7% at 15micromol/l and 3.8% at 600micromol/l. The lower limit of quantification was determined to 5micromol/l and precision was 5.5% and 5.0% for sulfadoxine and sulfamethoxazole, respectively.

Importance of pre-analytical factors contributing to measurement uncertainty, when determining sulfadoxine and sulfamethoxazole from capillary blood dried on sampling paper.

A bioanalytical method is developed and validated for determination of sulfadoxine (SD) and sulfamethoxazole (SM) in 100 microL capillary blood dried on sampling paper (Whatman 31ET Chr). SD and SM are extracted with 2000 microL perchloric acid and the liquid phase is loaded onto ENV+ solid-phase extraction columns. SD, SM, and the internal standard are separated on a Purospher STAR RP-18 liquid chromatography column (150 x 4.6 mm) with a mobile phase consisting of acetonitrile-sodium acetate buffer pH 5.2, I = 0.1 (33:67, v/v). Analytes are detected with UV at 256 nm. Lower limit of quantitation is 5 micromol/L, where precisions are 4.2% and 3.9% for SD and SM, respectively. Three brands of sampling papers have been compared with respect to absorption properties, extraction recoveries, and variations. Punching out dried blood spots (DBS) instead of cutting spots into strips prior to extraction has been evaluated by examining precision and accuracy of SD and SM determinations. Importance of uniformity of types of sampling paper, sampling volume and biological matrix, benefit of punching out discs from DBS, and impact on absorption properties of different brands of sampling papers are discussed. Avoiding pre-analytical errors whenever possible results in concentrations determined being more accurate and precise.

Enantioselective and nonlinear intestinal absorption of eflornithine in the rat.

This study aimed to investigate if the absorption of the human African trypanosomiasis agent eflornithine was stereospecific and dose dependent after oral administration. Male Sprague-Dawley rats were administered single doses of racemic eflornithine hydrochloride as an oral solution (750, 1,500, 2,000, or 3,000 mg/kg of body weight) or intravenously (375 or 1,000 mg/kg of body weight). Sparse blood samples were obtained for determination of eflornithine enantiomers by liquid chromatography with evaporative light-scattering detection (lower limit of quantification [LLOQ], 83 microM for 300 microl plasma). The full plasma concentration-time profile of racemic eflornithine following frequent sampling was determined for another group of rats, using a high-performance liquid chromatography-UV method (LLOQ, 5 microM for 50 microl plasma). Pharmacokinetic data were analyzed in NONMEM for the combined racemic and enantiomeric concentrations. Upon intravenous administration, the plasma concentration-time profile of eflornithine was biphasic, with marginal differences in enantiomer kinetics (mean clearances of 14.5 and 12.6 ml/min/kg for L- and D-eflornithine, respectively). The complex absorption kinetics were modeled with a number of transit compartments to account for delayed absorption, transferring the drug into an absorption compartment from which the rate of influx was saturable. The mean bioavailabilities for L- and D-eflornithine were 41% and 62%, respectively, in the dose range of 750 to 2,000 mg/kg of body weight, with suggested increases to 47% and 83%, respectively, after a dose of 3,000 mg/kg of body weight. Eflornithine exhibited enantioselective absorption, with the more potent L-isomer being less favored, a finding which may help to explain why clinical attempts to develop an oral treatment have hitherto failed. The mechanistic explanation for the stereoselective absorption remains unclear.

Determination of eflornithine enantiomers in plasma, by solid-phase extraction and liquid chromatography with evaporative light-scattering detection.

A bioanalytical method for determination of eflornithine (DFMO) in 1000 microL human plasma has been developed and validated. DFMO and the internal standard (IS) were analysed by liquid chromatography with evaporative light-scattering detection (ELSD). Separation was performed on a Chirobiotic TAG (250 mm x 4.6 mm) column with ethanol (99.5%):0.01 mol/L acetic acid-triethylamine buffer at the rate of 25:75% (v/v) with flow rate of 1.0 mL/min. For d-DFMO in plasma the inter-assay precision was 6.5% at 75 micromol/L, 6.6% at 375 micromol/L and 5.8% at 750 micromol/L. For l-DFMO in plasma the inter-assay precision was 10.4% at 75 micromol/L, 6.5% at 375 micromol/L and 5.0% at 750 micromol/L. The lower limit of quantification (LLOQ) was determined to 25 micromol/L where the precision was 4.3% and 5.7%, respectively.

Automated solid-phase extraction method for the determination of piperaquine in capillary blood applied onto sampling paper by liquid chromatography.

A bioanalytical method for the determination of piperaquine in 100 microL blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Blood spots were cut into small pieces prior to addition of 0.3M perchloric acid, acetonitrile and phosphate buffer containing an internal standard. The liquid phase was loaded onto a mixed phase cation-exchange (MPC) solid-phase extraction column. Piperaquine and the internal standard were analysed by liquid chromatography and separated on a Chromolith Performance (100 mm x 4.6 mm) column with acetonitrile:phosphate buffer pH 2.5, I = 0.1 (8:92, v/v) at the flow of 3.5 mL/min. The UV detection was performed at 345 nm. The intra-assay precision was 12.0% at 0.150 microM, 7.3% at 1.25 microM and 7.3% at 2.25 microM. The inter-assay precision was 1.8% at 0.150 microM, 5.2% at 1.25 microM and 2.8% at 2.25 microM. The lower limit of quantification (LLOQ) was determined to 0.050 microM where the precision was 14.7%.

A prospective and randomized study, "SVEA," comparing effects of three methods for delayed breast reconstruction on quality of life, patient-defined problem areas of life, and cosmetic result.

During the last 30 years, many methods for delayed breast reconstruction have been described. There is a lack of prospective randomized trials comparing reconstruction methods. The present study (SVEA), conducted 1995 to 1996, describes the impacts of three methods: the lateral thoracodorsal flap, the latissimus dorsi flap, and the pedicled transverse rectus abdominis muscle flap (TRAM), on important areas of life, patients' perception of cosmetic result, and quality of life. Questionnaires were completed before randomization and at 6 and 12 months postoperatively. The preoperative questionnaire concerned the impact of breast loss and expectations on reconstruction. Follow-up questionnaires dealt with satisfaction with cosmetic result and impact on important areas of life. A health-related quality-of-life questionnaire (SF-36) was completed at all points of assessment. A total of 75 of 87 randomized patients underwent breast reconstruction: 16 patients with the lateral thoracodorsal flap, 30 with the latissimus dorsi flap, and 29 with the TRAM flap. The majority were very satisfied with the cosmetic result. Most women reported improvements in important areas of life, and quality of life in terms of "social functioning" and "mental health" increased significantly after the reconstruction. The latissimus dorsi flap and TRAM flap scored significantly higher as compared with the lateral thoracodorsal flap for similarity with the contralateral breast and reduced problems in social situations. No differences between irradiated and nonirradiated patients were found. All methods were considered to produce good cosmetic results and improvements in patient-defined problem areas of life and quality of life. No negative effects were recorded. Thus, irrespective of method, breast reconstruction is a valuable tool for the mastectomized woman to cope with problems in everyday life.

The lateral thoracodorsal flap in breast reconstruction: a comparison between two plastic surgical centres.

In a retrospective study the results of breast reconstruction with 146 lateral thoracodorsal flaps operated on during the period 1991-94 at the Departments of Plastic Surgery at the Sahlgrenska University Hospital in Göteborg and the Stockholm Söder Hospital were evaluated. The median age of the 135 patients was 51 years (range 37-74). About two thirds of the patients were also operated on on the opposite side at the same time; and this was done more often in Stockholm, which partly explains the longer operating time in Stockholm. Perioperative bleeding (median 100 ml, range 25-400) was similar in both groups. Median postoperative bleeding, which was measured only in Stockholm, was 300 ml (range 30-1000). Seromas were seen only in Göteborg where postoperative drains were rarely used. The rate of early complications (infection and partial necrosis) were higher in Stockholm where more flaps were raised in irradiated tissue. We conclude that the results were similar in the two centres, and the thoracodorsal flap gives good results. The incidence of early morbidity could be reduced by excluding patients who had been irradiated, by inserting drains, and by giving antibiotic prophylaxis.

Clinical follow up of the lateral thoracodorsal flap in breast reconstruction: comparative evaluation from two plastic surgical centres.

A follow up study that compared the results from two plastic surgical centres comprised 95 patients after breast reconstruction with the lateral thoracodorsal (LTD) flap combined with either a smooth or textured saline-filled implant. It was conducted a median of 39 months (range 15-67) after the reconstruction. We investigated the size of the reconstructed breast and LTD flap, symmetry of the breasts, orientation of the mastectomy scar, and the rate of capsular contracture, which were similar in the two centres. However, according to the modified Baker classification and applanation tonometry reconstructions with smooth-surfaced implants and drainage of the implant pocket resulted in softer breasts (p = 0.03). The LTD flap technique in breast reconstruction was a good choice, particularly for women who required a minor breast reconstruction, and the technique can be recommended for suitable patients.

A prospective randomised study (named SVEA) of three methods of delayed breast reconstruction. Study design, patients' preoperative problems and expectations.

A randomised study named SVEA to compare three methods for delayed breast reconstruction was initiated in 1994. The methods were: the lateral thoracodorsal flap, the latissimus dorsi flap, and the pedicled transverse rectus abdominis muscle flap. In the present paper we describe the study design, primary endpoints, and inclusion and exclusion criteria. Results from preoperative questionnaires about problems after mastectomy and expectations of the reconstruction are presented. The preoperative questionnaires, completed before randomisation, included a health related quality of life questionnaire, the SF-36, and a questionnaire concerning the impact of breast loss and expectations of the reconstruction. A total of 87 patients have been randomised in SVEA and 30 patients have been followed up outside the randomised study, comprising a reference group. The results from preoperative questionnaires, completed by 106 of the total 117 women, showed that they encountered many problems after mastectomy, primarily about feeling mutilated and being bothered socially. They held high expectations on the breast reconstruction in these areas. The women in the present study did not differ from Swedish women in general regarding health related quality of life, with two exceptions: the study sample scored lower on physical functioning and mental health.

Clinical experience with the lateral thoracodorsal flap in breast reconstruction.

The lateral thoracodorsal flap is a local fasciocutaneous flap used for breast reconstruction. From 1986 to 1994 the authors used this flap in 157 breast reconstructions in 152 patients. The patients who had been treated with postmastectomy radiotherapy (N = 40) had significantly more (p < 0.01) early complications, such as necrosis and infection. Irradiated patients also required more operations due to secondary complications, such as capsular contracture (p < 0.05). A patient questionnaire (response rate, 91%) was sent to 121 patients and showed that, although a majority of patients felt the reconstructed breast was less sensitive to touch and harder than the contralateral breast, they still regarded the reconstructed breast as a natural part of themselves. More than 90% of patients did not regret the reconstruction and 80% would recommend it to a friend.

alpha4 integrin-dependent eotaxin induction of bronchial hyperresponsiveness and eosinophil migration in interleukin-5 transgenic mice.

We investigated the roles of eosinophil infiltration and activation induced by the eosinophil-selective chemokine eotaxin, and of the expression of eosinophil alpha4 and beta2 integrins in causing bronchial hyperresponsiveness (BHR) in interleukin (IL)-5 CBA/Ca transgenic mice. These mice did not show BHR, despite the presence of some eosinophils in the lungs. Intratracheal mouse recombinant eotaxin (3 micrograms) did not induce BHR in wild-type mice. In IL-5 transgenic mice, eotaxin (3 and 5 micrograms) increased responsiveness at 24 h and increased eosinophils in bronchoalveolar lavage (BAL) fluid by 9.4- and 14-fold by 24 h, respectively, together with augmentation of eosinophil peroxidase activity and eosinophil infiltration in the airway submucosa. Using flow cytometry, the expression of alpha4, CD11b, and CD18 was upregulated in BAL, but not in blood, eosinophils. A rat anti-alpha4 antibody inhibited eotaxin-induced BHR and eosinophil migration and activation, but an anti-CD11b antibody had no significant effects on BHR. A combination of both antibodies was more effective. IL-5 and eotaxin synergize in the induction of BHR and airway eosinophilia, effects that are dependent on the induction of eosinophil alpha4 integrin. Expression of BHR depends on the recruitment and activation of eosinophils.

The inflammatory reaction in elective flap surgery.

The inflammatory response in three different flap procedures was investigated by measuring the preoperative and postoperative levels of C-reactive protein, leukocyte count, and body temperature. Patients scheduled for delayed breast reconstruction were operated on with the lateral thoracodorsal flap, the latissimus dorsi flap, or the pedicled TRAM flap. All patients received 2 gm of intravenous cloxacillin for antibiotic prophylaxis and 1 gm of paracetamol four times a day as basic treatment for postoperative pain. Within each treatment group, significant postoperative changes in C-reactive protein levels, leukocyte count, and body temperature were noted when compared with preoperative values. The highest C-reactive protein level (130 mg/ml) was found in the TRAM group on the third postoperative day. The kinetic pattern of C-reactive protein was similar for the latissimus dorsi flap and lateral thoracodorsal flap procedures, but the maximum C-reactive protein levels were significantly lower, 74 and 44 mg/ml respectively. Small (0.5 to 0.9 degrees C) but significant differences in body temperature were also noted on the second and third postoperative day. The TRAM flap group had the highest, the latissimus dorsi flap group intermediate, and the lateral thoracodorsal flap group the lowest value. The postoperative C-reactive protein levels seem to reflect the extent of the surgical trauma.

Metabolism in myocutaneous flaps studied by in situ microdialysis.

The aim of this investigation was to follow the metabolism of myocutaneous flaps using microdialysis. After subcutaneous implantation of a microdialysis catheter into the flap tissue, serial samples were collected and changes in composition of the extracellular fluid assessed. Ten women underwent reconstructions with transverse rectus abdominis myocutaneous (TRAM) or latissimus dorsi flaps. Glucose, glycerol, and lactate concentrations were measured in the flaps and compared with those in adipose control tissue located over the hip. A transient rise in glucose was observed initially in the flaps. The glycerol concentration also increased significantly, and remained increased for approximately 12 hours after the operation. The lactate concentration changed in the same direction, but stayed elevated in the flap tissue during the entire investigation (24 hours). Neither of these parameters changed to the same extent in the control tissue. In one of the patients a haematoma developed postoperatively in the flap. This incident was accompanied by a sharp decline in glucose, and marked additional increases in glycerol and lactate concentrations. It may well be that this "metabolic pattern" in flap tissue signals threatening flap ischaemia. If so, this new microdialysis technique may be useful as a postoperative surveillance tool in myocutaneous flap surgery.

Serum creatine kinase in fasciocutaneous and musculocutaneous flap surgery.

The pre- and postoperative serum levels of creatine kinase (CK) were analyzed in 56 patients operated with three different breast reconstructive procedures. Thirteen patients were operated with the lateral thoracodorsal flap (LTD), 23 by the latissimus dorsi flap (LD), and 20 by the pedicled transverse rectus abdominis musculocutaneous (TRAM) flap. Nineteen patients in the LD and TRAM groups had received postmastectomy radiotherapy. The operations caused a significant rise in the serum levels of CK, and the highest levels were found on the first postoperative day: LTD, 5.4 microkat per liter; LD, 8.1 microkat per liter; and TRAM, 7.3 microkat per liter. There was no significant difference in CK levels between the groups. The irradiated patients did not show increased CK levels when compared with nonirradiated patients.

Hamartoma of the breast: surgical treatment and reconstruction. Case report.

Hamartoma of the breast is a rare and usually benign tumour with a variable growth pattern. Slowly increasing breast asymmetry is the most common clinical finding. Smaller hamartomas are often diagnosed on mammography. Simple enucleation and secondary expansion of the breast tissue are often sufficient to restore breast symmetry but sometimes, as in the two cases presented, reconstruction is necessary.