Demographic, clinical, and service-use characteristics related to the clinician's recommendation to transition from child to adult mental health services.

PURPOSE: The service configuration with distinct child and adolescent mental health services (CAMHS) and adult mental health services (AMHS) may be a barrier to continuity of care. Because of a lack of transition policy, CAMHS clinicians have to decide whether and when a young person should transition to AMHS. This study describes which characteristics are associated with the clinicians' advice to continue treatment at AMHS. METHODS: Demographic, family, clinical, treatment, and service-use characteristics of the MILESTONE cohort of 763 young people from 39 CAMHS in Europe were assessed using multi-informant and standardized assessment tools. Logistic mixed models were fitted to assess the relationship between these characteristics and clinicians' transition recommendations. RESULTS: Young people with higher clinician-rated severity of psychopathology scores, with self- and parent-reported need for ongoing treatment, with lower everyday functional skills and without self-reported psychotic experiences were more likely to be recommended to continue treatment. Among those who had been recommended to continue treatment, young people who used psychotropic medication, who had been in CAMHS for more than a year, and for whom appropriate AMHS were available were more likely to be recommended to continue treatment at AMHS. Young people whose parents indicated a need for ongoing treatment were more likely to be recommended to stay in CAMHS. CONCLUSION: Although the decision regarding continuity of treatment was mostly determined by a small set of clinical characteristics, the recommendation to continue treatment at AMHS was mostly affected by service-use related characteristics, such as the availability of appropriate services.

Protocol for the development and validation procedure of the managing the link and strengthening transition from child to adult mental health care (MILESTONE) suite of measures.

BACKGROUND: Mental health disorders in the child and adolescent population are a pressing public health concern. Despite the high prevalence of psychopathology in this vulnerable population, the transition from Child and Adolescent Mental Health Services (CAMHS) to Adult Mental Health Services (AMHS) has many obstacles such as deficiencies in planning, organisational readiness and policy gaps. All these factors contribute to an inadequate and suboptimal transition process. A suite of measures is required that would allow young people to be assessed in a structured and standardised way to determine the on-going need for care and to improve communication across clinicians at CAMHS and AMHS. This will have the potential to reduce the overall health economic burden and could also improve the quality of life for patients travelling across the transition boundary. The MILESTONE (Managing the Link and Strengthening Transition from Child to Adult Mental Health Care) project aims to address the significant socioeconomic and societal challenge related to the transition process. This protocol paper describes the development of two MILESTONE transition-related measures: The Transition Readiness and Appropriateness Measure (TRAM), designed to be a decision-making aide for clinicians, and the Transition Related Outcome Measure (TROM), for examining the outcome of transition. METHODS: The TRAM and TROM have been developed and were validated following the US FDA Guidance for Patient-reported Outcome Measures which follows an incremental stepwise framework. The study gathers information from service users, parents, families and mental health care professionals who have experience working with young people undergoing the transition process from eight European countries. DISCUSSION: There is an urgent need for comprehensive measures that can assess transition across the CAMHS/AMHS boundary. This study protocol describes the process of development of two new transition measures: the TRAM and TROM. The TRAM has the potential to nurture better transitions as the findings can be summarised and provided to clinicians as a clinician-decision making support tool for identifying cases who need to transition and the TROM can be used to examine the outcomes of the transition process. TRIAL REGISTRATION: MILESTONE study registration: ISRCTN83240263 Registered 23-July-2015 - ClinicalTrials.gov NCT03013595 Registered 6 January 2017.

Skin grafts from genetically modified α-1,3-galactosyltransferase knockout miniature swine: A functional equivalent to allografts.

Burn is associated with a considerable burden of morbidity worldwide. Early excision of burned tissue and skin grafting of the resultant wound has been established as a mainstay of modern burn therapy. However, in large burns, donor sites for autologous skin may be limited. Numerous alternatives, from cadaver skin to synthetic substitutes have been described, each with varying benefits and limitations. We previously proposed the use of genetically modified (alpha-1,3-galactosyl transferase knockout, GalT-KO) porcine skin as a viable skin alternative. In contrast to wild type porcine skin, which has been used as a biologic dressing following glutaraldehyde fixation, GalT-KO porcine skin is a viable graft, which is not susceptible to loss by hyperacute rejection, and undergoes graft take and healing, prior to eventual rejection, comparable to cadaver allogeneic skin. In the current study we aimed to perform a detailed functional analysis of GalT-KO skin grafts in comparison to allogeneic grafts for temporary closure of full thickness wounds using our baboon dorsum wound model. Grafts were assessed by measurement of fluid loss, wound infection rate, and take, and healed appearance, of secondary autologous grafts following xenograft rejection. Comparison was also made between fresh and cryopreserved grafts. No statistically significant difference was identified between GalT-KO and allogeneic skin grafts in any of the assessed parameters, and graft take and function was not adversely effected by the freeze-thaw process. These data demonstrate that GalT-KO porcine grafts are functionally comparable to allogeneic skin grafts for temporary closure of full thickness wounds, and support their consideration as an alternative to cadaver allogeneic skin in the emergency management of large burns.

Resonant coupling in the heteronuclear alkali dimers for direct photoassociative formation of X(0,0) ultracold molecules.

Promising pathways for photoassociative formation of ultracold heteronuclear alkali metal dimers in their lowest rovibronic levels (denoted X(0,0)) are examined using high-quality ab initio calculations of potential energy curves currently available. A promising pathway for KRb, involving the resonant coupling of the 2(1)Pi and 1(1)Pi states just below the lowest excited asymptote (K(4s) + Rb(5p(1/2))), is found to occur also for RbCs and less promisingly for KCs also. The resonant coupling of the 3(1)Sigma(+) and 1(1)Pi states, also just below the lowest excited asymptote, is found to be promising for LiNa, LiK, and LiRb and less promising for LiCs and KCs. Direct photoassociation to the 1(1)Pi state near dissociation appears promising in the final dimers NaK, NaRb, and NaCs, although detuning more than 100 cm(-1) below the lowest excited asymptote may be required.

Neuropathic and cerebrovascular correlates of hearing loss in Fabry disease.

Fabry disease, OMIM 301500, is a progressive multisystem storage disorder due to the deficiency of alpha-galactosidase A (GALA). Neurological and vascular manifestations of this disorder with regard to hearing loss have not been analysed quantitatively in large cohorts. We conducted a retrospective cross sectional analysis of hearing loss in 109 male and female patients with Fabry disease who were referred to and seen at the Clinical Center of the National Institutes of Health, Bethesda, MD, USA on natural history and enzyme replacement study protocols. There were 85 males aged 6-58 years (mean 31 years, SD 13) and 24 females aged 22-72 years (mean 42 years, SD 12). All patients underwent a comprehensive audiological evaluation. In addition, cerebral white matter lesions, peripheral neuropathy, and kidney function were quantitatively assessed. HL(95), defined as a hearing threshold above the 95th percentile for age and gender matched normal controls, was present in 56% [95% CI (42.2-67.2)] of the males. Prevalence of HL(95) was lower in the group of patients with residual GALA enzyme activity compared with those without detectable activity (33% versus 63%) HL(95) was present in the low-, mid- and high-frequency ranges for all ages. Male patients with HL(95) had a higher microvascular cerebral white matter lesion load [1.4, interquartile range (IQR) 0-30.1 +/- versus 0, IQR 0-0], more pronounced cold perception deficit [19.4 +/- 5.5 versus 13.5 +/- 5.5 of just noticeable difference (JND) units] and lower kidney function [creatinine: 1.6 +/- 1.2 versus 0.77 +/- 0.2 mg/dl; blood urea nitrogen (BUN): 20.1 +/- 14.1 versus 10.3 +/- 3.28 mg/dl] than those without HL(95) (P < 0.001). Of the females, 38% had HL(95). There was no significant association with cold perception deficit, creatinine or BUN in the females. Word recognition and acoustic reflexes analyses suggested a predominant cochlear involvement. We conclude that hearing loss involving all frequency regions significantly contributes to morbidity in patients with Fabry disease. Our quantitative analysis suggests a correlation of neuropathic and vascular damage with hearing loss in the males. Residual GALA activity appears to have a protective effect against hearing loss.

Vertical transmission of hepatitis C virus in a cohort of 2,447 HIV-seronegative pregnant women: a 24-month prospective study.

BACKGROUND: Mother to infant transmission of hepatitis C virus (HCV) has been extensively studied in mothers with human immunodeficiency virus (HIV) infection, whereas fewer data are available on the vertical HCV transmission in HIV-negative women. METHODS: Between January 1995 and June 1997, 78 consecutive HCV-positive/HIV-negative women with their offspring entered this prospective study aimed to define the prevalence of and risk factors for HCV vertical transmission. Risk factors for HCV were carefully sought, and HCV viral load and genotype were determined in all positive mothers. The infants were tested for alanine aminotransferase (ALT) and HCV-RNA at birth and at 4, 8, 12, 18, and 24 months of age. RESULTS: Eight of 60 (13.3%) infants born to HCV-RNA positive mothers acquired HCV infection, but only 2 (3,3%) were still infected by the end of follow-up. Infants' genotypes matched that of the mothers. ALT levels were in the normal range in all study subjects throughout the follow-up. High maternal viral load (P < 0.05), possession of HCV risk factors (P < 0.004), and history of blood transfusion (P < 0.05) were associated with increased risk of HCV vertical transmission. CONCLUSIONS: This long-term prospective study shows that, although vertical transmission from HIV-negative mothers occurs in 13% of cases, there is a high rate of spontaneous viral clearance (75%). High maternal viral load and mothers belonging to HCV risk categories were the only variables predictive of the vertical transmission.

Auditory dysfunction in Stickler syndrome.

OBJECTIVES: To characterize the natural history and possible mechanisms of hearing loss in Stickler syndrome (OMIM 108300; or hereditary progressive arthro-ophthalmopathy) and to determine if the auditory phenotype is a useful discriminating feature for the differential diagnosis of this group of disorders. DESIGN: Multifamily study. SETTING: Outpatient audiology and otolaryngology clinics at the Warren Grant Magnuson Clinical Center of the National Institutes of Health, Rockville, Md. SUBJECTS: Forty-six affected individuals from 29 different families segregating Stickler syndrome. INTERVENTIONS: Clinical audiologic and otolaryngological examinations were performed on all individuals, including pure-tone audiometry, speech audiometry, and middle ear immittance testing. Otoacoustic emissions, auditory brainstem response, infrared video electronystagmography, and temporal bone computed tomography were performed on a subset of participants. RESULTS: The hearing loss was most often sensorineural in adults, and approximately 28 (60%) of the 46 adult patients had 2 or more thresholds greater than the corresponding 95th percentile values for an age-matched, otologically normal population. The hearing loss most often affected high frequencies (4000-8000 Hz) and was generally no more progressive than that due to age-related hearing loss. Type A(D) tympanograms (classification using the Jerger model), indicating hypermobile middle ear systems, were observed in 21 (46%) of the 46 affected individuals. Computed tomography of the temporal bones revealed no inner ear malformations in 19 affected individuals. CONCLUSIONS: The hypermobile middle ear systems observed in ears with normal-appearing tympanic membranes represent a novel finding for Stickler syndrome and are likely to be a useful diagnostic feature for this disorder. The overall sensorineural hearing loss in type I Stickler syndrome is typically mild and not significantly progressive. It is less severe than that reported for types II and III Stickler syndrome linked to COL11A2 (OMIM 120290) and COL11A1 (OMIM 120280) mutations, respectively, or the closely related Marshall syndrome. This difference will be a useful discriminatory feature in the differential diagnosis of this group of disorders.

Dominant modifier DFNM1 suppresses recessive deafness DFNB26.

More than 50% of severe childhood deafness is genetically determined, approximately 70% of which occurs without other abnormalities and is thus termed nonsyndromic. So far, 30 nonsyndromic recessive deafness loci have been mapped and the defective genes at 6 loci, DFNB1, DFNB2, DFNB3, DFNB4, DFNB9 and DNFB21, have been identified, encoding connexin-26 (ref. 3), myosin VIIA (ref. 4), myosin XV (ref. 5), pendrin, otoferlin and alpha-tectorin, respectively. Here we map a new recessive nonsyndromic deafness locus, DFNB26, to a 1.5-cM interval of chromosome 4q31 in a consanguineous Pakistani family. A maximum lod score of 8.10 at theta=0 was obtained with D4S1610 when only the 8 affected individuals in this family were included in the calculation. There are seven unaffected family members who are also homozygous for the DFNB26-linked haplotype and thus are non-penetrant. A dominant modifier, DFNM1, that suppresses deafness in the 7 nonpenetrant individuals was mapped to a 5.6-cM region on chromosome 1q24 with a lod score of 4.31 at theta=0 for D1S2815.

Primary peritoneal carcinoma: a report of twelve cases and a review of the literature.

Primary peritoneal carcinoma (PPC) is rare tumor histologically identical to epithelial ovarian carcinoma (EOC); it is differentiated from EOC based on the extent of gross ovarian involvement and microscopic invasion of the cortex. We report 12 cases of PPC which were diagnosed in our Department during a 9-year period. Total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy were performed in 9 patients, while 3 underwent only explorative laparotomy with bilateral salpingo-oophorectomy. All patients were treated with postoperative platinum-based chemotherapy. After a median follow-up of 42 months, only 5 patients are alive without disease. PPC is a rare tumor currently managed in the same way as ovarian cancer. Primary debulking surgery and chemotherapy represent the cornerstones of treatment. Considering the limited number of patients with PPC, no definitive conclusion can be drawn concerning the prognostic factors for survival.

[Reconstructive surgery in oncological gynecology. Our experience]. / La chirurgia ricostruttiva in ginecologia oncologica. Nostra esperienza.

BACKGROUND: Personal experience in reconstructive surgery for gynecologic oncology is reported. These techniques are playing, during last years, a leading role since it is necessary to assure a good treatment and top quality lifestyle to oncologic patients. METHODS: A retrospective study was made on 357 major surgical treatments for neoplastic disease at the IInd Clinic of Obstetrics and Gynecology, University of Bari. For each disease the technique, the length of surgery and possible complications have been reported. In total 50 reconstructive procedures on 357 surgical interventions for gynecologic neoplastic disease (14%) were made. RESULTS: There was no rate of mortality, and the complications were found only in 5 patients (10%). CONCLUSIONS: Reconstructive surgery can increase surgical radicality, improve the quality of life of the patients and reduce the postoperative complications.

[Combination of cisplatin and vinorelbin in the neoadjuvant treatment of locally advanced cervical carcinoma. Phase II study]. / Associazione di cisplatino e vinorelbina nel trattamento neoadiuvante del cancro cervicale localmente avanzato. Studio di fase II.

BACKGROUND: Aim of this phase II study is to evaluate the cytoreductive efficacy and the toxicity of a regimen consisting of cisplatin and vinorelbine as neoadjuvant chemotherapy for three cycles every 21 days in patients with locally-advanced cervical carcinoma. METHODS: Between April 1996 and December 1998, 33 untreated patients with locally-advanced cervical carcinoma received neoadjuvant chemotherapy with cisplantino 80 mg/m2 (day 1) plus vinorelbine 25 mg/m2 (day 1 and 8). Within 28 days from completion of chemotherapy patients in complete or partial response were submitted to radical hysterectomy plus pelvic lymphadenectomy. RESULTS: Twenty-six patients (79%) were submitted to radical surgery; four patients were not submitted to surgery because of poor response to treatment, two for anesthesiological contraindications and one refused surgery. The combination of cisplatin plus vinorelbine in this phase II study induced a clinical overall response rate of 90%, with 8 pathologic complete response (24%). CONCLUSIONS: The association of cisplatin and vinorelbine as neoadjuvant chemotherapy in locally-advanced cervical carcinoma showed to be safe and effective with a low cost and poor toxicity.

Splenic involvement in ovarian cancer. Description of three cases.

Metastatic involvement of the spleen in patients with ovarian cancer is unusual. Solitary splenic metastasis in the absence of disseminated disease are rare. Three cases of advanced ovarian cancer with splenic involvement are reported. The splenectomy was adopted in two patients.

There is substantial nuclear and cellular disintegration before detectable phosphatidylserine exposure during the camptothecin-induced apoptosis of HL-60 cells.

BACKGROUND: An early sign of apoptosis in many cells is the appearance of phosphatidylserine (PS) on the outside of the plasma membrane, whilst the cells still retain the ability to exclude DNA-binding molecules such as propidium iodide and 7-aminoactinomycin D (7-AAD). The protein annexin V binds preferentially to PS and has often been used to monitor the early phase of apoptosis. There have been some conflicting results concerning whether annexin V binds to camptothecin (CAM)-treated HL-60 cells, a commonly used model for apoptosis. We investigated the effects of culturing HL-60 cells for up to 8 h with a range of CAM concentrations. METHODS: We used flow cytometry to measure cellular light scatter, annexin V-FITC binding, and 7-AAD uptake, and DNA content after fixation and permeabilization. We also used microscopy to examine the morphology of cells (both unsorted and sorted according to their light scatter) after cytocentrifugation. RESULTS: We found that CAM caused the rapid appearance of low light scatter apoptotic bodies. Even among cells with "normal" light scatter, there was widespread DNA cleavage and nuclear fragmentation by 3 h. The percentage of apoptotic bodies peaked at about 4 h and it was only afterward that annexin V binding could be detected to both intact cells and to apoptotic bodies. When they first appeared, the intact annexin V+ cells had S-phase DNA content. CONCLUSIONS: During CAM-induced apoptosis of HL-60 cells, the external exposure of PS can either precede or follow DNA cleavage, which suggests that PS exposure is not always an indicator of early apoptosis.

[Ursodeoxycholic acid in the therapy of chronic hepatitis after treatment with interferon]. / L'acido ursodesossicolico nella terapia delle epatiti croniche virali dopo trattamento con interferone.

UNLABELLED: Interferon alfa is to day the only therapy of proven benefit for the treatment and control of chronic hepatitis C. Therefore only 25% of patients receive from it a sustained biochemical and serological response; often when the treatment is stopped there is a flare up of ALT and reappearance of HCV-RNA in the serum. The most common schedule is 6 MU t.i w. for twelve months; after this there is no codified treatment for relapse prevention. The aim of this study was to evaluate if ursodeoxycholic acid (UDCA) administration after a cycle of IFN therapy was able to prevent relapse of the disease. METHODS: We studied 36 patients whose mean age was 31.5 +/- 5.7 affected by chronic hepatitis C and treated with IFN alpha for one year. Only twenty of them received an end term therapy response and were therefore enrolled in a double blind study with two arms: Arm A treated with UDCA 300 mg b. i. d. for twelve months and Arm B treated with placebo. ALT value and HVC-RNA levels were evaluated at baseline, during and after treatment. RESULTS: Patients treated with UDCA showed a lower percentage of relapse in comparison with patients treated with placebo. CONCLUSIONS: This effect was probably due to a double mechanism: the first of biochemical type because a reduction in the intrahepatic concentration of hydrophobic biliary acids, the second immunological due to a lower expression of HLA class I and II antigens.

[Efficacy of insulin Lispro in the control of late postprandial hypoglycemia: comparison with regular human insulin]. / Efficacia dell'insulina lispro nel controllo della ipoglicemia postprandiale tardiva: confronto con la insulina umana regolare.

BACKGROUND: Lispro insulin a recently developed analogue of human insulin, is more rapidly adsorbed and has a lower duration of activity as compared with regular insulin. This implies a glycemic profile closer to the physiologic one with a reduction of early post-prandial hyperglycemic peak and of drop in late postprandial glycemia. This results in a reduction of mild or severe hypoglycemia occurring during treatment with regular human insulin. METHODS: This was designed to evaluate the efficacy of Lispro insulin in the metabolic control in subjects treated with regular insulin who were prone to late hypoglycemia. Fifteen subjects, 6 males and 9 females, range of age 18-54 years with insulin-dependent diabetes mellitus (IDDM) have been studied. These subjects were treated with regular insulin at meals plus intermediate in the evening. Regular insulin was substituted with Lispro insulin. The glycemic profile and HbA1c have been evaluated at determined intervals. Also body mass index and the number of hypoglycemic events during treatment were recorded. Significance of differences was assessed by paired Student's "t"-test. RESULTS: Lispro insulin reduced the peaks of early postprandial hyperglycemic peak and, in particular, the late glycemic drop, Lispro insulin reduced also HbA1c levels thus suggesting a better metabolic control. Moreover the number of hypoglycemic events was significantly reduced. CONCLUSIONS: In conclusion, Lispro insulin is safe and more efficient than regular human insulin.

Hetero-domain interactions as a mechanism for the regulation of connexin channels.

Previous studies have shown that chemical regulation of connexin43 (Cx43) depends on the presence of the carboxyl terminal (CT) domain. A particle-receptor (or "ball-and-chain") model has been proposed to explain the mechanism of gating. We tested whether the CT region behaved as a functional domain for other members of the connexin family. The pH sensitivity of wild-type and Ct-truncated connexins was quantified by use of electrophysiological and optical techniques and the Xenopus oocyte system. The CT domain of Cx45 had no role in pH regulation, although a partial role was shown for Cx37 and Cx50. A prominent effect was observed for Cx40 and Cx43. In addition, we found that the CT domain of Cx40 that was expressed as a separate fragment rescued the pH sensitivity of the truncated Cx40 (Cx40tr), which was in agreement with a particle-receptor model. Because Cx40 and Cx43 often colocalize and possibly heteromerize, we tested the pH sensitivity of Cx40tr when coexpressed with the CT domain of Cx43 (hetero-domain interactions). We found that the CT domain of Cx43 enhanced the pH sensitivity of Cx40tr; similarly, the CT domain of Cx40 restored the pH sensitivity of the truncated Cx43. In addition, the CT domain of Cx43 granted insulin sensitivity to the otherwise insulin-insensitive Cx26 or Cx32 channels. These data show that the particle-receptor model is preserved in Cx40 and the regulatory domain of one connexin can specifically interact with a channel formed by another connexin. Hetero-domain interactions could be critical for the regulation of heteromeric channels.

Cochlear outer hair cell electromotility can provide force for both low and high intensity distortion product otoacoustic emissions.

It is generally believed that the force for the otoacoustic emission (OAE) generation is provided by a mechanism of electromotility, observed in isolated cochlear outer hair cells (OHCs). OHC electromotility is resistant to several ototoxic reagents, it does not depend on ATP hydrolysis, but it can be blocked by specific sulfhydryl reagents: p-chloromercuriphenylsulfonic acid (pCMPS) and p-hydroxymercuriphenylsulfonic acid (pHMPS). We have used these reagents to test whether they also affect OAE. Application of pCMPS and pHMPS on the round window membrane of anesthetized guinea pigs produced a dose-dependent inhibition of the cubic (2F1-F2) distortion product OAE (DPOAE). The inhibition developed progressively from high to low frequencies, reflecting the diffusion of the drugs through the cochlear compartment. The effect of pCMPS and pHMPS was different from the effects of furosemide and lethal anoxia, which impair cochlear function but do not block OHC electromotility. pHMPS suppressed DPOAE completely at all sound intensities tested (45-80 dB SPL), whereas furosemide or lethal anoxia caused DPOAE to disappear at low-level stimulation (45-60 dB SPL) only. Our results suggest that the OHC electromotility might provide the force for DPOAE generation not only at low, but also at high stimulus intensities.

Prevalence and mechanisms of hearing loss in patients with resistance to thyroid hormone.

Hearing impairment was anecdotally reported in resistance to thyroid hormone (RTH), a condition caused by mutations in the beta-thyroid hormone receptor (beta TR) gene. Because of its ontogenic distribution in the cochlea, the beta TR may have a pivotal role in the development of auditory function. To assess the prevalence and mechanisms of hearing impairment in RTH, 82 RTH-positive (RTH+) patients and 55 unaffected relatives (RTH-) underwent systematic audiological examination, including puretone and speech reception thresholds, and tests studying middle ear (tympanometry and acoustic reflexes), cochlear (otoacoustic emissions), and retrocochlear integrity (brain stem auditory evoked potentials). Significant hearing loss was present in 21% of RTH+ patients vs. none in RTH- patients. More RTH+ patients had abnormal tympanometry (34% vs. 12%) and abnormal acoustic reflexes (39% vs. 19%). Isolated conductive deficit was found in 7 of 17 RTH+ patients with hearing loss, isolated sensorineural deficit in 7 cases, and mixed deficit in 3 cases. Cochlear dysfunction was found in 50% of all RTH+ patients, with or without hearing loss. Retrocochlear function was normal. No morphological cochlear abnormalities were detected on computed tomography of the temporal bone. In conclusion, hearing loss is a significant problem in RTH, with an equal frequency of conductive (probably related to the frequent ear infections) and sensorineural deficits. Abnormal otoacoustic emissions suggest that the mutant beta TR has a specific negative impact on cochlear function.

Use of four-colour flow cytometry to evaluate conjugate formation between human peripheral blood mononuclear cells and tumour target cells.

A four-colour flow cytometry technique is described for the determination of the number and phenotype of conjugate-forming mononuclear effector cells from human blood. The discriminatory power of previously described techniques was improved by labelling the effector cells with antibodies that simultaneously identified natural killer (NK), T, and myeloid cells and by labelling the tumour target cells with fluorescein octadecyl ester (FOE), so that cell conjugates could be differentiated from nonbinding effector cells more effectively than by the use of light scatter. The simultaneous characterisation of the three classes of conjugate-forming cells within a heterogeneous human peripheral blood mononuclear cell (PBMC) population made it possible to investigate conjugate formation by each subpopulation without purification, or semipurification (e.g., by monocyte removal), of the subpopulations. The binding of PBMCs to K562 and RPMI 1788 targets was examined. Binding for all three PBMC subpopulations was optimal at 37 degrees C and was negligible at 0 degree C (except for some T cell binding to RPMI 1788 cells). At 37 degrees C, maximum binding was essentially achieved by 10 min. Sodium azide inhibited the majority of the conjugation by NK and T cells, and that inhibition could be removed by washing the cells prior to conjugation, whereas azide had a negligible effect on the binding by monocytes. It appears that effective conjugation by human peripheral blood NK and T lymphocytes requires the operation of an energy-dependent process, differentiating it from conjugation by monocytes.

Natural killer cells and CD56+ T cells in the blood of multiple myeloma patients: analysis by 4-colour flow cytometry.

The blood of multiple myeloma patients was examined for non-MHC-restricted cytotoxic lymphocytes. Four colour flow cytometry was used to phenotype the cells within a light scatter gate large enough to include all lymphocytes. NK and T cells were identified using CD16, CD56, and CD3 antibodies, and myeloid cells with CD13 and CD14 antibodies. Three subpopulations of NK cells and 3 subpopulations of CD16+ or CD56+ T cells were enumerated. The CD56+ NK and T cells were also examined with CD69, CD25, and anti-HLA-DR antibodies to assess their activation state. We found no evidence that either the percentage or the absolute number of any subpopulation of the NK cells or CD56+ T cells correlated with disease activity. Neither did we find any significant abnormalities in the numbers of activated CD56+ NK or T cells. We conclude that it is unlikely that circulating non-MHC-restricted cytotoxic lymphocytes are responsible for maintaining disease stability in myeloma patients with indolent disease.