Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

The role of subchondral bone, and its histomorphology, on the dynamic viscoelasticity of cartilage, bone and osteochondral cores.

OBJECTIVE: Viscoelastic properties of articular cartilage have been characterised at physiological frequencies. However, studies investigating the interaction between cartilage and subchondral bone and the influence of underlying bone histomorphometry on the viscoelasticity of cartilage are lacking. METHOD: Dynamic Mechanical Analysis (DMA) has been used to quantify the dynamic viscoelasticity of bovine tibial plateau osteochondral cores, over a frequency sweep from 1 to 88 Hz. Specimens (approximately aged between 18 and 30 months) were neither osteoarthritic nor otherwise compromised. A maximum nominal stress of 1.7 MPa was induced. Viscoelastic properties of cores have been compared with that of its components (cartilage and bone) in terms of the elastic and viscous components of both structural stiffness and material modulus. Micro-computed tomography scans were used to quantify the histomorphological properties of the subchondral bone. RESULTS: Opposing frequency-dependent loss stiffness, and modulus, trends were witnessed for osteochondral tissues: for cartilage it increased logarithmically (P < 0.05); for bone it decreased logarithmically (P < 0.05). The storage stiffness of osteochondral cores was logarithmically frequency-dependent (P < 0.05), however, the loss stiffness was typically frequency-independent (P > 0.05). A linear relationship between the subchondral bone plate (SBP) thickness and cartilage thickness (P < 0.001) was identified. Cartilage loss modulus was linearly correlated to bone mineral density (BMD) (P < 0.05) and bone volume (P < 0.05). CONCLUSION: The relationship between the subchondral bone histomorphometry and cartilage viscoelasticity (namely loss modulus) and thickness, have implications for the initiation and progression of osteoarthritis (OA) through an altered ability of cartilage to dissipate energy.

Maternal engineered nanomaterial inhalation during gestation alters the fetal transcriptome.

BACKGROUND: The integration of engineered nanomaterials (ENM) is well-established and widespread in clinical, commercial, and domestic applications. Cardiovascular dysfunctions have been reported in adult populations after exposure to a variety of ENM. As the diversity of these exposures continues to increase, the fetal ramifications of maternal exposures have yet to be determined. We, and others, have explored the consequences of ENM inhalation during gestation and identified many cardiovascular and metabolic outcomes in the F1 generation. The purpose of these studies was to identify genetic alterations in the F1 generation of Sprague-Dawley rats that result from maternal ENM inhalation during gestation. Pregnant dams were exposed to nano-titanium dioxide (nano-TiO2) aerosols (10 ± 0.5 mg/m3) for 7-8 days (calculated, cumulative lung deposition = 217 ± 1 µg) and on GD (gestational day) 20 fetal hearts were isolated. DNA was extracted and immunoprecipitated with modified chromatin marks histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 27 tri-methylation (H3K27me3). Following chromatin immunoprecipitation (ChIP), DNA fragments were sequenced. RNA from fetal hearts was purified and prepared for RNA sequencing and transcriptomic analysis. Ingenuity Pathway Analysis (IPA) was then used to identify pathways most modified by gestational ENM exposure. RESULTS: The results of the sequencing experiments provide initial evidence that significant epigenetic and transcriptomic changes occur in the cardiac tissue of maternal nano-TiO2 exposed progeny. The most notable alterations in major biologic systems included immune adaptation and organismal growth. Changes in normal physiology were linked with other tissues, including liver and kidneys. CONCLUSIONS: These results are the first evidence that maternal ENM inhalation impacts the fetal epigenome.

Raman-shifted wavelength-selectable pulsed fiber laser with high repetition rate and high pulse energy in the visible.

A high-pulse-energy, diffraction-limited, wavelength-selectable, visible source, based on Raman frequency shifting of a frequency-doubled Yb-doped fiber laser, has been studied. The relative length-scaling laws of Raman gain and self-phase modulation push the design towards short fiber lengths with large core size. It is experimentally demonstrated that the Raman clean-up effect in a graded-index multi-mode fiber is not sufficient to obtain diffraction-limited beam quality in the short fiber length. Thus, a large-core photonic crystal fiber is used to maintain diffraction-limited performance and output pulse energies of ~1 µJ, at a 1-MHz repetition rate and 1.3-ns pulse-width are successfully achieved. This step-tunable visible source should find applications in photoacoustic microscopy.

Thulium-fiber-laser-pumped, high-peak-power, picosecond, mid-infrared orientation-patterned GaAs optical parametric generator and amplifier.

We report a high-peak-power, picosecond, mid-infrared optical parametric generator (OPG) and optical parametric amplifier (OPA) based on orientation-patterned gallium arsenide pumped by a thulium:fiber master-oscillator-power-amplifier employing direct diode-seeded amplification. An OPG tuning range of 2550-2940 nm (signal) and 5800-8300 nm (idler) is demonstrated with peak powers as high as 3 (signal) and 2 kW (idler), and with pulse energies of 0.26 and 0.16 µJ, respectively. When seeded with a 0.6 cm-1 linewidth tunable Cr:ZnSe laser, the OPA idler linewidth is narrowed to 1.4 cm-1 and a small-signal parametric gain of 60 dB is achieved. A maximum peak power of 13.3 (signal) and 3.2 kW (idler) is obtained at an overall conversion efficiency of 36%. The corresponding maximum pulse energies for the signal and idler are 1.07 and 0.26 µJ, respectively.

Fatigue strength of bovine articular cartilage-on-bone under three-point bending: the effect of loading frequency.

BACKGROUND: The objective of this study was to determine the influence of loading frequency on the failure of articular cartilage-on-bone specimens under three-point bending. METHODS: In this study, cyclic three-point bending was used to introduce failure into cartilage-on-bone specimens at varying loading frequencies. Sinusiodally varying maximum compressive loads in the range 40-130 N were applied to beam-shaped cartilage-on-bone specimens at frequencies of 1, 10, 50 and 100 Hz. RESULTS: The number of cycles to failure decreased when loading frequency increased from normal and above gait (1 and 10 Hz) to impulsive loading frequencies (50 and 100 Hz). It was found that 67 and 27% of the specimens reached run-out at loading of 10,000 cycles at frequencies of 1 and 10 Hz, respectively. However, 0% of the specimens reached run-out at loading frequencies of 50 and 100 Hz. CONCLUSION: The results indicate that increasing the loading frequency reduces the ability of specimens to resist fracture during bending. The findings underline the importance of the loading frequency concerning the failure of articular cartilage-on-bone and it may have implications in the early onset of osteoarthritis.

Effect of the variation of loading frequency on surface failure of bovine articular cartilage.

BACKGROUND: Mechanical loading of synovial joints can damage the articular cartilage surface and may lead to osteoarthritis. It is unknown if, independent of load, frequency alone can cause failure in cartilage. This study investigated the variation of articular cartilage surface damage under frequencies associated with normal, above normal and traumatic loading frequencies. METHOD: Cartilage on bone, obtained from bovine shoulder joints, was tested. Damage was created on the cartilage surface through an indenter being sinusoidally loaded against it at loading frequencies of 1, 10 and 100 Hz (i.e., relevant to normal, above normal and up to rapid heel-strike rise times, respectively). The frequencies were applied with a maximum load in the range 60-160 N. Surface cracks were marked with India ink, photographed and their length measured using image analysis software. RESULTS: Surface damage increased significantly (P < 0.0001) with frequency throughout all load ranges investigated. The dependence of crack length, c, on frequency, f, could be represented by, c=A(log10(f))2+B(log10(f))+Dc=A(log10(f))2+B(log10(f))+D where A = 0.006 ± 0.23, B = 0.62 ± 0.23 and D = 0.38 ± 0.51 mm (mean ± standard deviation). CONCLUSION: The increase in crack length with loading frequency indicated that, increased loading frequency can result in cartilage becoming damaged. The results of this study have implications in the early stages of osteoarthritis.

Fiber-laser-pumped, high-energy, mid-IR, picosecond optical parametric oscillator with a high-harmonic cavity.

We demonstrate the generation of high-energy, mid-IR, picosecond pulses in a high-harmonic-cavity optical parametric oscillator (OPO) that has a relatively compact cavity with a length that is a small fraction of that required to match the pump repetition rate. The OPO, based on an MgO-doped periodically poled LiNbO3 crystal, is pumped by a fiber master-oscillator-power-amplifier system employing direct amplification and delivering 11-µJ, 150-ps pulses at 1035 nm. For a 1.554-m-long OPO cavity, resonating near-infrared signal pulses with a repetition rate that is the 193rd harmonic of the 1-MHz pump are demonstrated. The mid-infrared idler output pulses, tunable from 2300 nm to 3500 nm, are generated at a 1-MHz repetition rate and have energies as high as 1.5 µJ.

Responsible corporate change: detecting and managing employee stress.

BACKGROUND: All 120 health and safety inspectors employed by the New Zealand regulatory agency had their jobs disestablished during a restructuring process and were required to undergo an assessment process with tight time frames. AIMS: To report on psychological morbidity during the transition to change. METHODS: The Hospital Anxiety and Depression Scale (HADS) questionnaire was emailed to all 120 current inspectors to measure levels of anxiety (HAD-A) and depression (HAD-D). A score of <7 is in the normal range, scores of between 8 and 10 are suggestive of an anxiety (HADS-A) or mood (HADS-D) disorder and a score of >11 is indicative of a clinical disorder. RESULTS: Replies were received from 36% (43) of the inspectors. Of the 40 usable responses, 47% (19) and 55% (22), respectively, had HAD-A and HAD-D scores greater than the case cut-off. Only 28% (11) and 15% (6), respectively, had scores that would be considered normal. CONCLUSIONS: The high scores evident in this sample are comparable to those found in patients with serious psychopathology. Change managers should recognize that the onus for primary prevention lies with the organization, in this case designing an assessment process that takes place over a reasonable time frame. They should also realize the requirement for the active monitoring of stress.

Chewing, rumen pool characteristics, and lactation performance of dairy cows fed 2 concentrations of a corn wet-milling coproduct with different forage sources.

We used a novel corn wet-milling coproduct [CMP; approximately 70% dry matter, 28% crude protein, 36% neutral detergent fiber (NDF), and 18% nonstructural carbohydrates] in diets formulated to contain 18.4% forage NDF, 17.4% crude protein, 20.2% starch, and 3.7% sugar. Six primiparous, rumen-cannulated Jersey cows were assigned to a 6 × 6 Latin square design with a 2 × 3 factorial arrangement of treatments. Diets were formulated to contain 20 and 30% CMP with 3 forage sources [corn silage (CS) and 40.5% NDF, CS replaced with 10% alfalfa hay (AH) and 45.0% NDF, or CS replaced with 7% grass hay (GH) and 67.4% NDF], with each providing 18.4% forage NDF in the diet. Total-tract digestibilities of NDF, N, and organic matter were not affected by treatment. Similarly, no treatment effects were detected for kinetics of NDF disappearance in situ from CMP or respective forage source or for N disappearance in situ from CMP. Grass hay increased total and liquid pool size of rumen contents compared with AH (by 3.2 and 3.0kg, respectively). Total time spent chewing increased in cows fed GH by over 35min compared with those fed AH, partially due to a trend for increased minutes spent ruminating. Mean particle size of rumen contents also tended to be higher in the GH (0.55mm) than AH (0.69mm) diets. No effects on production of milk or milk components were detected, but dry matter intake (DMI) tended to decrease when CMP increased from 20 to 30%. Gross feed efficiency (fat-corrected milk/DMI) tended to be greater when cows were fed AH and GH compared with CS and was greater for AH than GH diets. In diets containing low starch, increasing CMP from 20 to 30% potentially maintained similar fat-corrected milk production with lower DMI. However, more consideration also should be given to interactions among forages with respect to fill, digestion, and passage of fiber with increased inclusion rates of CMP.

Rigid intramedullary nail fixation of femoral fractures in adolescents: what evidence is available?

BACKGROUND: Femoral fracture in adolescents is a significant injury. It is generally agreed that operative fixation is the treatment of choice, and rigid intramedullary nail fixation is a treatment option. However, numerous types of rigid nails to fix adolescent femoral fractures have been described. Hence, the aim of this paper was to collate and evaluate the available evidence for managing diaphyseal femoral fractures in adolescents using rigid intramedullary nails. MATERIALS AND METHODS: A literature search was undertaken using the healthcare database website ( http://www.library.nhs.uk/hdas ). Medline, CINAHL, Embase, and the Cochrane Library databases were searched to identify prospective and retrospective studies of rigid intramedullary nail fixation in the adolescent population. RESULTS: The literature search returned 1,849 articles, among which 51 relevant articles were identified. Of these 51 articles, 23 duplicates were excluded, so a total of 28 articles were reviewed. First-generation nails had a high incidence of limb length discrepancy (Küntscher 5.8 %, Grosse-Kempf 9 %), whilst second-generation nails had a lower incidence (Russell-Taylor 1.7 %, AO 2.6 %). Avascular necrosis was noted with solid Ti nails (2.6 %), AO femoral nails (1.3 %) and Russell-Taylor nails (0.85 %). These complications have not been reported with the current generation of nails. CONCLUSIONS: Rigid intramedullary nail fixation of femoral fractures in adolescents is a useful procedure with good clinical results. A multiplanar design and lateral trochanteric entry are key to a successful outcome of titanium alloy nail fixation.

Diode-pumped femtosecond solid-state waveguide laser with a 4.9 GHz pulse repetition rate.

We report on the first demonstration of a passively mode-locked, diode-pumped, monolithic Yb:glass channel waveguide laser that incorporates a semiconductor saturable absorber mirror. Stable and self-starting mode-locking is achieved in a Fabry-Perot cavity configuration producing a pulse repetition rate up to 4.9 GHz. The shortest pulse duration of 740 fs is generated with 30 mW of average output power at a center wavelength of 1058 nm. A maximum output power of 81 mW is produced during mode-locking with corresponding pulse duration of 800 fs.

Use of algae or algal oil rich in n-3 fatty acids as a feed supplement for dairy cattle.

Fish oil is used as a ration additive to provide n-3 fatty acids to dairy cows. Fish do not synthesize n-3 fatty acids; they must consume microscopic algae or other algae-consuming fish. New technology allows for the production of algal biomass for use as a ration supplement for dairy cattle. Lipid encapsulation of the algal biomass protects n-3 fatty acids from biohydrogenation in the rumen and allows them to be available for absorption and utilization in the small intestine. Our objective was to examine the use of algal products as a source for n-3 fatty acids in milk. Four mid-lactation Holsteins were assigned to a 4×4 Latin square design. Their rations were supplemented with 1× or 0.5× rumen-protected (RP) algal biomass supplement, 1× RP algal oil supplement, or no supplement for 7 d. Supplements were lipid encapsulated (Balchem Corp., New Hampton, NY). The 1× supplements provided 29 g/d of docosahexaenoic acid (DHA), and 0.5× provided half of this amount. Treatments were analyzed by orthogonal contrasts. Supplementing dairy rations with rumen-protected algal products did not affect feed intake, milk yield, or milk component yield. Short- and medium-chain fatty acid yields in milk were not influenced by supplements. Both 0.5× and 1× RP algae supplements increased daily milk fat yield of DHA (0.5 and 0.6±0.10 g/d, respectively) compared with 1× RP oil (0.3±0.10 g/d), but all supplements resulted in milk fat yields greater than that of the control (0.1±0.10g/d). Yield of trans-18:1 fatty acids in milk fat was also increased by supplementation. Trans-11 18:1 yield (13, 20, 27, and 15±3.0 g/d for control, 0.5× RP algae, 1× RP algae, and 1× RP oil, respectively) was greater for supplements than for control. Concentration of DHA in the plasma lipid fraction on d 7 showed that the DHA concentration was greatest in plasma phospholipid. Rumen-protected algal biomass provided better DHA yield than algal oil. Feeding lipid-encapsulated algae supplements may increase n-3 content in milk fat without adversely affecting milk fat yield; however, preferential esterification of DHA into plasma phospholipid may limit its incorporation into milk fat.

Design of a surgical instrument for removing bone to provide screw access to a spinal fusion cage.

A surgical instrument to aid implantation of a range of lumbar spinal fusion cages has been developed. Once the cage is in position, the entrance to screw holes is partially blocked by the edge of the vertebral body. In order to insert fixation screws to secure the cage between the vertebrae, some part of the blocking edge has to be removed. Rongeurs are currently being used, but they can be time consuming and have the disadvantage that they may remove more bone than is necessary and may cause damage to the fusion cage if not used with care. In addition, access around some of the screw holes may be difficult. The aim of this instrument was to overcome these shortcomings. This paper describes the design of a surgical instrument for cutting edges from vertebral bodies. The development and evaluation of concept designs are presented and discussed. Potential risks were considered and modifications were performed on the selected concept. Functional prototypes were manufactured and tested on sheep lumbar vertebrae. The results showed that the newly designed cutting instrument functions as required and removes the required amount of bone from the vertebral body edge.

INDOLE-3-CARBINOL EXERTS SEX-SPECIFIC EFFECTS IN MURINE COLITIS.

Due to the severe adverse effects that can accompany conventional therapies for Crohn's disease, the search for natural complementary therapies has increased dramatically in recent years. Indole-3-carbinol (I3C), a constituent of cruciferous vegetables, possesses anti-inflammatory properties; however, its effects on intestinal inflammation have yet to be evaluated. To test the hypothesis that I3C dampens intestinal inflammation, C57Bl/6 mice were treated with I3C and exposed to 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce colitis. Several parameters of disease severity and inflammation were subsequently evaluated. I3C dampened the disease severity, as indicated by decreased body weight loss and decreased severity of clinical signs. Interestingly, this effect was observed in female but not male mice, which displayed a trend towards exacerbated colitis. Differential effects were observed in the profiles of cytokine production, as the production of pro-inflammatory cytokines was increased in males. The sex-specific effect of I3C in TNBS-induced colitis is a novel finding and warrants further investigation since this is a common dietary compound and is also available commercially.

Effect of side holes in cervical fusion cages: a finite element analysis study.

The objective of this study was to investigate the effect of side holes on the predicted von Mises stress levels in cervical spinal fusion cages subjected to compressive loading. Models with between zero and ten side holes were developed. Finite element analysis (FEA) was used to simulate compression of the cage, made from the polymer PEEK (polyetheretherketone), between two adjacent vertebrae. The analyses were validated by experimental tests. In all of the models, the von Mises stress was highest at the cage-vertebrae interface with peak stresses of between 14 and 18 MPa. Increasing the Young's modulus of the vertebrae from 12 to 30 GPa increased the peak stress on average by 29 per cent. The stresses in the models were lower than the compressive strength of PEEK (118 MPa), and are well within the PEEK fatigue strength reported (60 MPa at 10 million cycles). This study suggests that the number of side holes had a negligible effect on the stress distribution within the cage; the stress magnitudes were fairly constant across all of the models and did not change substantially with the number of holes. Hence, a cervical cage with side holes is unlikely to fail in compression.

Validation of the WHOQOL-BREF quality of life questionnaire for use with medical students.

PURPOSE: The purpose of the present study was to validate the use of the abbreviated version of the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire with medical students. METHODS: A sample of 274 medical students in their fourth and fifth years of study (80% response rate) completed the WHOQOL-BREF instrument. The four-domain factor structure of the questionnaire was tested using confirmatory factor analysis. A variety of other tests of reliability and validity were conducted. RESULTS: Goodness-of-fit indices from the confirmatory factor analysis were acceptable, and the factor structure of the WHOQOL-BREF was confirmed in this sample. Reliability was good, but three of the items showed strong ceiling effects. CONCLUSION: The WHOQOL-BREF is valid to use with medical students to assess health-related quality of life. Some items, such as those inquiring about pain and medication, may not be suitable for medical students or young people in general.

The contribution of personal audio system use and commuting by bus on daily noise dose.

BACKGROUND: For many young people, exposure to music from personal audio system use may represent a significant component of daily noise dose. Moreover, there is increasing concern for the hearing of those who listen at high volumes. The purpose of this study was to determine the noise levels experienced on commuter buses, and to investigate how these impact on the volume-setting behavior of young adult personal audio system users. METHODS: A questionnaire was used to probe transport use, personal audio system-listening behaviors and the extent of understanding about noise-induced hearing loss. The influence of bus noise on volume-setting behavior was determined by measuring, in a lab setting, the sound-level preferences of participants when listening to their favorite song, a generic song, or a podcast in the absence and presence of various levels of bus noise, simulated using output-adjusted recordings made of bus noise. Statistical analysis was conducted using analysis of variance. RESULTS: While the bus noise itself was below 85 dB Leq, as the sound level of the buses increased, so did the percentage of commuters who were found to exceed the equivalent of 8 hours of exposure at 85 dB Leq. IMPLICATIONS: Investment in buses with lower noise levels or the use of noise-canceling or noise-occluding headphones would help to reduce the likelihood of noise-induced hearing loss for bus commuters.

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39.

The ligand for CD40 has been recently identified as a 39-kd protein, gp39, expressed on the surface of activated CD4+ T helper cells (Th). In vitro, soluble CD40 and anti-gp39 have been shown to block the ability of Th to activate B cells, suggesting that gp39-CD40 interactions are important to T cell-dependent B cell activation. Here it is shown that in vivo administration of anti-gp39 dramatically reduced both primary and secondary humoral immune responses to erythrocytes and soluble protein antigens without altering responses to the T-independent type II antigen, trinitrophenyl-Ficoll. Treatment of mice for 4 d with anti-gp39 inhibited the anti-sheep red blood cell (SRBC) response for at least 3 wk and inhibited the expression of all immunoglobulin isotypes in secondary responses to the protein antigen, keyhole limpet hemocyanin. To examine the direct effect of anti-gp39 on Th function, SRBC-immune Th cells from anti-gp39-treated mice were adoptively transferred and shown to be fully capable of providing help. These results suggest that anti-gp39 treatment does not cause Th deletion or anergy. Anti-gp39 may mediate its profound immunosuppressive effects on humoral immunity by blocking gp39-CD40 interactions. Moreover, these studies establish gp39-CD40 as an important receptor-ligand pair for the targeting of therapeutic antibodies to control thymus-dependent humoral responses.

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. I. In vivo expression of CD40 ligand, cytokines, and antibody production delineates sites of cognate T-B cell interactions.

T-B cell interactions have a central role in the development of antibody responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activation. The cytokines produced by activated Th cells have a regulatory role in B cell differentiation. In this study, we investigated, using immunohistochemical techniques, the in vivo time course and localization of gp39 expression and cytokine production in relation to the specific antibody production. Both the immunization with keyhole limpet hemocyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP)-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around the terminal arterioles (TA). Incidentally, gp39+ Th cells were found in the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary immunization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that activated T cells are involved in TI-2 antibody responses. Analysis of the in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL-4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) developed according to similar kinetics as observed for gp39+ cells. IL-2-PC and IL-4-PC were present in higher frequencies as were IFN-gamma-PC in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma, suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine-PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on activated Th cells after immunization with TD and TI-2 antigens. Furthermore, the time course and compartmentalization of gp39+ expression, cytokine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentiation occur in the outer-PALS and around the TA of the spleen.